High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

Translocation of HSP47 and generation of mitochondrial reactive oxygen species in human neuroblastoma SK-N-SH cells following electron and X-ray irradiation.

Generation of mitochondrial reactive oxygen species (ROS), lipid peroxidation, 4-hydroxy-2-nonenal, and heat-shock protein (HSP) 47 after electron and X-ray irradiations were detected in the human neuroblastoma cell line SK-N-SH. After 10 Gy electron irradiation and 15 Gy X-ray irradiation, mitochondrial ROS production and lipid peroxidation were significantly increased. Additionally, we observed a significant increase in the levels of HSP47 after 3 and 10 Gy electron irradiation as well as 15 Gy X-ray irradiation. Furthermore, myristoylation and farnesylation were increased after 10 Gy electron and 15 Gy X-ray irradiations. We found that the level of HSP47 increased in the mitochondria after 10 Gy electron and 15 Gy X-ray irradiations. HSP47 coexisted with myristoylation and farnesylation. Furthermore, HSP47 overexpression increased mitochondrial ROS production. These results suggest that HSP47 plays an important role in mitochondria and induces mitochondrial ROS production in SK-N-SH cells.

Evolutionary conservation and post-translational control of S-adenosyl-L-homocysteine hydrolase in land plants.

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.

Post-translational modifications of protein in response to ionizing radiation.

Based on central dogma of genetics, protein is the embodiment and executor of genetic function, post-translational modifications (PTMs) of protein are particularly important and involved in almost all aspects of cell biology and pathogenesis. Studies have shown that ionizing radiation (IR) alters gene expression much more profoundly and a broad variety of cell-process pathways, lots of proteins are modified and activated. Our understanding of the protein in response to ionizing radiation is steadily increasing. Among the various biological processes known to induce radioresistance, PTMs have attracted marked attention in recent years. The present review summarizes the latest knowledge about how PTMs response to ionizing radiation and pathway analysis were conducted. The data provided insights into biological effects of IR and contributing to the development of novel IR-based strategies.

ßarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1.

Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the ß2-adrenergic-ßarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, ßarrestin-1 (ßarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether ßarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice ßarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that ßarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of ßarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, ßarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, ßarr1 is an important regulator of double strand break repair, and disruption of the ßarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

Repurposing Protein Degradation for Optogenetic Modulation of Protein Activities.

Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control the intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellular signal-regulated kinase (ERK) pathway, and a constitutively active form of MEK (CA MEK), a positive regulator of the same pathway. Kinetics study showed that light-induced protein stabilization could be achieved within 30 min of blue light stimulation. GLIMPSe enables target-independent optogenetic control of protein activities and therefore minimizes the systematic variation embedded within different photoactivatable proteins. Overall, GLIMPSe promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.

Radiation-Induced CXCL12 Upregulation via Histone Modification at the Promoter in the Tumor Microenvironment of Hepatocellular Carcinoma.

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24- cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.

Radiation-promoted CDC6 protein stability contributes to radioresistance by regulating senescence and epithelial to mesenchymal transition.

Ionizing radiation (IR) is a conventional cancer therapeutic, to which cancer cells develop radioresistance with exposure. The residual cancer cells after radiation treatment also have increased metastatic potential. The mechanisms by which cancer cells develop radioresistance and gain metastatic potential are still unknown. In this study acute IR exposure induced cancer cell senescence and apoptosis, but after long-term IR exposure, cancer cells exhibited radioresistance. The proliferation of radioresistant cells was retarded, and most cells were arrested in G0/G1 phase. The radioresistant cells simultaneously showed resistance to further IR-induced apoptosis, premature senescence, and epithelial to mesenchymal transformation (EMT). Acute IR exposure steadily elevated CDC6 protein levels due to the attenuation of ubiquitination, while CDC6 overexpression was observed in the radioresistant cells because the insufficiency of CDC6 phosphorylation blocked protein translocation from nucleus to cytoplasm, resulting in subcellular protein accumulation when the cells were arrested in G0/G1 phase. CDC6 ectopic overexpression in CNE2 cells resulted in apoptosis resistance, G0/G1 cell cycle arrest, premature senescence, and EMT, similar to the characteristics of radioresistant CNE2-R cells. Targeting CDC6 with siRNA promoted IR-induced senescence, sensitized cancer cells to IR-induced apoptosis, and reversed EMT. Furthermore, CDC6 depletion synergistically repressed the growth of CNE2-R xenografts when combined with IR. The study describes for the first time cell models for IR-induced senescence, apoptosis resistance, and EMT, three major mechanisms by which radioresistance develops. CDC6 is a novel radioresistance switch regulating senescence, apoptosis, and EMT. These studies suggest that CDC6highKI67low represents a new diagnostic marker of radiosensitivity, and CDC6 represents a new therapeutic target for cancer radiosensitization.

Protective effect of rutin against brain injury induced by acrylamide or gamma radiation: role of PI3K/AKT/GSK-3ß/NRF-2 signalling pathway.

This study was designed to evaluate the effect of rutin on PI3K/AKT-signalling in case of acrylamide or γ-radiation-induced neurotoxicity. To induce brain damage, animals were received acrylamide (25 mg/kg b.wt./orally/day) or 5 Gy of γ-radiation exposure accompanied with an administration of rutin (200 mg/kg b.wt./orally/day). Our data revealed that, compared to acrylamide or γ-radiation, rutin activated PI3K/AKT/GSK-3ß/NRF-2-pathway through increased protein levels of p-PI3K, p-AKT and p-GSK-3ß and up-regulated the expression of NRF-2. This was achieved by modulating MDA, GST, IL-1ß, IL-6 and reduced the interference of ROS with IGF-1 and NGF stimulating the PI3K/AKT-signaling. Furthermore, histopathological examinations of brain tissues showed that rutin has modulated tissue architecture after acrylamide or γ-radiation induced tissue damage. It could be concluded that rutin provides protection effect against acrylamide or γ-radiation-induced neurotoxicity via activation of the PI3K/AKT/GSK-3ß/NRF-2-pathway by altering the phosphorylation state through its ability to scavenge free radicals generation, modulating gene expression and its anti-inflammatory effects.

Effects of 1950 MHz radiofrequency electromagnetic fields on Aß processing in human neuroblastoma and mouse hippocampal neuronal cells.

Alzheimer's disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cognitive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aß), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aß plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aß processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aß processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aß peptide, was not significantly different between RF-EMF-exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observations suggest that RF-EMF exposure may not have a significant physiological effect on Aß processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aß processing with long-term and continuous exposure.

Post-illumination cellular effects of photodynamic treatment.

Increased interest in clinical application of photodynamic therapy (PDT) in various medical fields poses a demand for better understanding of processes triggered by photo-treatment. Most of the work on PDT performed so far has focused on the immediate effects of photo-treatment. It is generally accepted that cellular damage occurs during light exposure and within a short period thereafter. If cells are not killed during the PDT, they might recover, depending on the extent of the photo-induced damage. Little is known, however, about the relationship between the properties of photosensitizers (PSs) and the delayed consequences of PDT. The aim of this work was to investigate cellular responses to sub-lethal photodynamic treatment and how toxicogenic potency may be affected by molecular features of the PS. Results demonstrated that for cationic porphyrin-based PSs, lipophilicity is the main factor determining the fate of the cells in the 24-hour post-illumination period. PSs with amphiphilic properties initiated oxidative reactions that continued in the dark, long after light exposure, and caused suppression of metabolism and loss of cell viability with concomitant changes in electrophoretic mobility of proteins, including caspases. Apoptotic activity was not stimulated in the post-illumination period. This study demonstrated that in PDT mediated by amphiphilic cationic metalloporphyrin PSs, even when immediate photo-damage is relatively mild, destructive oxidative processes initiated during PDT continue in the absence of light to substantially impair metabolism, and that post-illumination protein modification may modify utilization of cell death pathways.

Palmitoylation is a prerequisite for dimerization-dependent raftophilicity of rhodopsin.

The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*-Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-ß-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-ß-cyclodextrin-resistant complex is 2:1. Next, we found that IgG-mediated Rh-Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG-cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.

Targeted Annotation of S-Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry.

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 µm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.

Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress.

Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions.

Secretome Signature Identifies ADAM17 as Novel Target for Radiosensitization of Non-Small Cell Lung Cancer.

PURPOSE: Ionizing radiation (IR) induces intracellular signaling processes as part of a treatment-induced stress response. Here we investigate IR-induced ADAM17 activation and the role of ADAM17-shed factors for radiation resistance in non-small cell lung cancer. EXPERIMENTAL DESIGN: Large-scale secretome profiling was performed using antibody arrays. Secretion kinetics of ADAM17 substrates was determined using ELISA across multiple in vitro and in vivo models of non-small cell lung cancer. Clonogenic survival and tumor xenograft assays were performed to determine radiosensitization by ADAM17 inhibition. RESULTS: On the basis of a large-scale secretome screening, we investigated secretion of auto- or paracrine factors in non-small cell lung cancer in response to irradiation and discovered the ADAM17 network as a crucial mediator of resistance to IR. Irradiation induced a dose-dependent increase of furin-mediated cleavage of the ADAM17 proform to active ADAM17, which resulted in enhanced ADAM17 activity in vitro and in vivo Genetic or pharmacologic targeting of ADAM17 suppressed IR-induced shedding of secreted factors, downregulated ErbB signaling in otherwise cetuximab-resistant target cells, and enhanced IR-induced cytotoxicity. The combined treatment modality of IR with the ADAM17 inhibitor TMI-005 resulted in a supra-additive antitumor response in vivo demonstrating the potential of ADAM17 targeting in combination with radiotherapy. CONCLUSIONS: Radiotherapy activates ADAM17 in non-small cell lung cancer, which results in shedding of multiple survival factors, growth factor pathway activation, and IR-induced treatment resistance. We provide a sound rationale for repositioning ADAM17 inhibitors as short-term adjuvants to improve the radiotherapy outcome of non-small cell lung cancer. Clin Cancer Res; 22(17); 4428-39. ©2016 AACR.

UV-dependent phosphorylation of COP9/signalosome in UV-induced apoptosis.

The COP9/signalosome (CSN) multi-protein complex regulates the activity of cullin-RING ubiquitin ligases (CRLs), including the DDB2 and CSA CRL4 ligases (CRL4DDB2 and CRL4CSA), which are involved in the repair of UV-induced DNA damages. In the present study, we demonstrated that the protein kinase ATM, a key component of the DNA damage response (DDR), phosphorylates CSN1 and CSN7a, two subunits of the CSN complex, in a UV-dependent manner. The phosphorylation of CSN1 on serine 474 was detected as early as 3 h after UV-exposure, peaked at 8 h and persisted until 48 h post-UV irradiation. Such a time course suggests a role in late DDR rather than in DNA repair. Consistently, overexpression of a phosphorylation-resistant S474A CSN1 mutant reduced UV-induced apoptosis. Thus, CSN1 appears to play a role not only in DNA repair but also in UV-induced apoptosis.

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4.

FBXW7 is a haploinsufficient tumor suppressor with loss-of-function mutations occurring in human cancers. FBXW7 inactivation causes genomic instability, but the mechanism remains elusive. Here we show that FBXW7 facilitates nonhomologous end-joining (NHEJ) repair and that FBXW7 depletion causes radiosensitization. In response to ionizing radiation, ATM phosphorylates FBXW7 at serine 26 to recruit it to DNA double-strand break (DSB) sites, whereas activated DNA-PKcs phosphorylates XRCC4 at serines 325/326, which promotes binding of XRCC4 to FBXW7. SCF(FBXW7) E3 ligase then promotes polyubiquitylation of XRCC4 at lysine 296 via lysine 63 linkage for enhanced association with the Ku70/80 complex to facilitate NHEJ repair. Consistent with these findings, a small-molecule inhibitor that abrogates XRCC4 polyubiquitylation reduces NHEJ repair. Our study demonstrates one mechanism by which FBXW7 contributes to genome integrity and implies that inactivated FBXW7 in human cancers could be a strategy for increasing the efficacy of radiotherapy.

UV Irradiation Triggers Cylindromatosis Translocation, Modification, and Degradation in a Proteasome-Independent Manner.

The tumor suppressor, cylindromatosis (CYLD), is a negative regulator of NF-κB signaling by removing lysine 63-linked ubiquitin chains from multiple NF-κB signaling components, including TRAF2, TRAF6, and NEMO. How CYLD itself is regulated, however, remains yet to be characterized. In this study, we present the first evidence that UV irradiation is able to induce CYLD translocation from the cytoplasm to microtubules and that the cytoskeleton-associated CYLD is subject to posttranslational modification and degradation in a proteasome-independent manner. By immunostaining, we found that CYLD displayed microtubule-like filament localization under ultraviolet (UV) irradiation. Further studies revealed that the cytoskeleton-associated CYLD underwent posttranslational modification, which in turn contributed to CYLD degradation in an unknown manner, distinct from proteasome-mediated degradation under normal conditions. Collectively, our data suggest that UV-induced CYLD degradation might serve as an underlying mechanism for UV-induced NF-κB pathway activation.

Histone H2AX Is Involved in FoxO3a-Mediated Transcriptional Responses to Ionizing Radiation to Maintain Genome Stability.

Histone H2AX plays a crucial role in molecular and cellular responses to DNA damage and in the maintenance of genome stability. It is downstream of ataxia telangiectasia mutated (ATM) damage signaling pathway and there is an emerging role of the transcription factor FoxO3a, a regulator of a variety of other pathways, in activating this signaling. We asked whether H2AX may feedback to FoxO3a to affect respective FoxO3a-dependent pathways. We used a genetically matched pair of mouse embryonic fibroblast H2AX(+/+) and H2AX(-/-) cell lines to carry out comprehensive time-course and dose-response experiments and to show that the expression of several FoxO3a-regulated genes was altered in H2AX(-)(/-) compared to H2AX(+/+) cells at both basal and irradiated conditions. Hspa1b and Gadd45a were down-regulated four- to five-fold and Ddit3, Cdkn1a and Sod2 were up-regulated 2-3-fold in H2AX(-/-) cells. Using the luciferase reporter assay, we directly demonstrated that transcriptional activity of FoxoO3a was reduced in H2AX(-/-) cells. FoxO3a localization within the nuclear phospho-ATM (Ser1981) foci in irradiated cells was affected by the H2AX status, as well as its posttranslational modification (phospho-Thr32). These differences were associated with genomic instability and radiosensitivity in H2AX(-/-) cells. Finally, knockdown of H2AX in H2AX(+/+) cells resulted in FoxO3a-dependent gene expression patterns and increased radiosensitivity that partially mimicked those found in H2AX(-/-) cells. Taken together, our data suggest a role for FoxO3a in the maintenance of genome integrity in response to DNA damage that is mediated by H2AX via yet unknown mechanisms.