Water molecules in protein-ligand interfaces. Evaluation of software tools and SAR comparison.

Targeting the interaction with or displacement of the 'right' water molecule can significantly increase inhibitor potency in structure-guided drug design. Multiple computational approaches exist to predict which waters should be targeted for displacement to achieve the largest gain in potency. However, the relative success of different methods remains underexplored. Here, we present a comparison of the ability of five water prediction programs (3D-RISM, SZMAP, WaterFLAP, WaterRank, and WaterMap) to predict crystallographic water locations, calculate their binding free energies, and to relate differences in these energies to observed changes in potency. The structural cohort included nine Bruton's Tyrosine Kinase (BTK) structures, and nine bromodomain structures. Each program accurately predicted the locations of most crystallographic water molecules. However, the predicted binding free energies correlated poorly with the observed changes in inhibitor potency when solvent atoms were displaced by chemical changes in closely related compounds.

Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson's disease.

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies.

Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling.

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.

AP-2 Is the Crucial Clathrin Adaptor Protein for CD4 Downmodulation by HIV-1 Nef in Infected Primary CD4+ T Cells.

HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, ß-COP, and ACOT8 for CD4 downmodulation in HIV-1-infected short hairpin RNA (shRNA)-expressing CD4(+) T cells and characterized direct interaction with Nef by Förster resonance energy transfer (FRET). Binding of lentiviral Nefs to CD4 and AP-2 was conserved, and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation.

Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834.

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways.

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-(2)H(3)]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

The Ebola virus glycoprotein (EBOV-GP) forms GP-containing microvesicles, so-called virosomes, which are secreted from GP-expressing cells. However, determinants of GP-virosome release and their functionality are poorly understood. We characterized GP-mediated virosome formation and delineated the role of the antiviral factor tetherin (BST2, CD317) in this process. Residues in the EBOV-GP receptor-binding domain (RBD) promote GP-virosome secretion, while tetherin suppresses GP-virosomes by interactions involving the GP-transmembrane domain. Tetherin from multiple species interfered with GP-virosome release, and tetherin from the natural fruit bat reservoir showed the highest inhibitory activity. Moreover, analyses of GP from various ebolavirus strains, including the EBOV responsible for the West African epidemic, revealed the most efficient GP-virosome formation by highly pathogenic ebolaviruses. Finally, EBOV-GP-virosomes were immunomodulatory and acted as decoys for EBOV-neutralizing antibodies. Our results indicate that GP-virosome formation might be a determinant of EBOV immune evasion and pathogenicity, which is suppressed by tetherin.

In vitro evaluation of magnetic resonance imaging at 3.0 tesla on clonogenic ability, proliferation, and cell cycle in human embryonic lung fibroblasts.

OBJECTIVES: We investigated the influence of magnetic resonance (MR) at 3.0 T on clonogenic ability, proliferation, and cell cycle in an embryonic human cell line. MATERIALS AND METHODS: Cells (human lung fibroblasts Hel 299) were exposed to the static magnetic field (3.0 T) of a magnetic resonance imager (MRI) and to a turbo spin echo sequence at 3.0 T within clinical limitations (specific absorption rate 0.92 W/kg). A special MR-compatible incubation system was used. A control group (sham-exposed) and a MRI group (exposed) were set up. We investigated 3 biologic endpoints: colony forming, cell cycle, and proliferation ability. The exposure time was 2 hours in each experiment. RESULTS: In the statistical analysis, none of these tests showed relevant differences between the exposed and sham-exposed group. CONCLUSIONS: No influences of the static field alone as well as a turbo spin echo sequence at 3.0 T on clonogenic ability, proliferation, or cell cycle in eugenic human lung fibroblasts were found.

Mutation analyses and association studies to assess the role of the presenilin-associated rhomboid-like gene in Parkinson's disease.

Presenilin-associated rhomboid-like (PARL), a serine protease located in the inner mitochondrial membrane, has been shown to genetically interact and process PTEN-induced putative kinase a protein known for its critical role in mitochondrial homeostasis and early-onset forms of Parkinson's disease (PD). The identification of a PD-associated variant in the PARL gene (p.Ser77Asn) led us to assess the relevance of PARL for PD pathogenesis using a mutation screening of the coding sequences and adjacent intronic sequences. We investigated 3 single nucleotide polymorphisms (rs3792589, rs13091, and rs3732581), a synonymous base substitution (Leu79Leu) and the previously described p.Ser77Asn mutation, which were subsequently screened in more than 2000 patients and controls. Not detecting the p.Ser77Asn mutation in our cohort, nor a robust association between variations in the PARL gene and PD, the role of disease causing genetic variants in the PARL gene could not be further substantiated in our samples. Our findings indicate that PARL mutations are a rare cause of PD and genetic variants are neither strong nor common risk factors in PD.

Maintaining physical activity during refeeding improves body composition, intestinal hyperpermeability and behavior in anorectic mice.

A role of gut-brain axis emerges in the pathophysiology of anorexia nervosa and maintaining adapted physical activity during refeeding remains discussed. We aimed to assess gastrointestinal protein metabolism and investigate the contribution of physical activity during refeeding in C57BL/6 mice with activity-based anorexia (ABA). ABA mice exhibited lower body weight and food intake with increase of lean mass/fat mass ratio and fat oxidation. Colonic permeability was increased in ABA. Ad libitum food access was then restored and ABA group was divided into two subgroups, with access to running wheel (ABA-PA) or not (ABA-NPA). After refeeding, fat free mass was completely restored only in ABA-PA. Colonic permeability was enhanced in ABA-NPA. Finally, muscle kynurenine conversion into kynurenic acid was lower in ABA-NPA who also exhibited altered behavior. Maintaining physical activity during refeeding may thus limit colonic hyperpermeability and improve behavior in anorectic mice.

Structural basis of the broadly neutralizing anti-interferon-α antibody rontalizumab.

Interferons-alpha (IFN-α) are the expressed gene products comprising thirteen type I interferons with protein pairwise sequence similarities in the 77-96% range. Three other widely expressed human type I interferons, IFN-ß, IFN-κ and IFN-ω have sequences 29-33%, 29-32% and 56-60% similar to the IFN-αs, respectively. Type I interferons act on immune cells by producing subtly different immune-modulatory effects upon binding to the extracellular domains of a heterodimeric cell-surface receptor composed of IFNAR1 and IFNAR2, most notably anti-viral effects. IFN-α has been used to treat infection by hepatitis-virus type C (HCV) and a correlation between hyperactivity of IFN-α-induced signaling and systemic lupus erythematosis (SLE), or lupus, has been noted. Anti-IFN-α antibodies including rontalizumab have been under clinical study for the treatment of lupus. To better understand the rontalizumab mechanism of action and specificity, we determined the X-ray crystal structure of the Fab fragment of rontalizumab bound to human IFN-α2 at 3Å resolution and find substantial overlap of the antibody and IFNA2 epitopes on IFN-α2.

Effects of transcranial direct current stimulation (tDCS) on executive functions: influence of COMT Val/Met polymorphism.

INTRODUCTION: Transcranial direct current stimulation (tDCS) is a frequently used technique to investigate healthy and impaired neuronal functions. Its modulatory effect on executive functions is of particular interest for understanding the mechanisms underlying integration of cognition and behavior. The key role of prefrontal dopamine function for executive functions suggest that differences of the Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene would interact with tDCS interventions in this domain. In this study, we hypothesized that the COMT Met allele homozygosity, associated with higher levels of prefrontal dopamine, would influence the effect of tDCS on higher-level executive functions. METHOD: Forty-six healthy subjects participated in a double-blind sham-controlled crossover study and underwent COMT genotyping. Anodal tDCS (20 min, 1 mA) to the left dorsolateral prefrontal cortex (dlPFC) or sham stimulation was applied during the performance of a parametric Go/No-Go (PGNG) test measuring sustained attention, response inhibition and cognitive flexibility as measured by set-shifting. RESULTS: In COMT Met/Met allele carrier anodal tDCS of the dlPFC was associated with a deterioration of set-shifting ability, which is assessed by the most challenging level of the PGNG. Without regard to the carrier status of the COMT Val158Met polymorphism no effects of anodal tDCS on executive functions could be determined. CONCLUSIONS: In line with the model of non-linear effects of l-dopa on cortical plasticity high dopaminergic prefrontal activity mediated by COMT Val158Met polymorphism predicts a detrimental effect of anodal tDCS on cognitive flexibility. Therefore, we suggest that the individual genetic profile may modulate behavioral effect of tDCS. More precise application of brain stimulation techniques according to the individual genetic patterns may support the development of personalized treatment approaches.

Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin-independent pathway.

BACKGROUND: Glutamine modulates duodenal protein metabolism in fasted healthy humans, but its effects in a fed state remain unknown. OBJECTIVE: We aimed to assess the effects of either glutamine or an isonitrogenous protein mixture on duodenal protein metabolism in humans in the fed state. DESIGN: Twenty-four healthy volunteers were randomly included in 2 groups. Each volunteer was studied on 2 occasions in a random order and received, during 5 h, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹; both groups) that mimicked a carbohydrate fed state or maltodextrins with glutamine (group 1) or an isonitrogenous (22.4 mg N · kg⁻¹ · h⁻¹) protein powder (group 2). Simultaneously, a continuous intravenous infusion of ¹³C-leucine and ²H5-phenylalanine (both 9 µmol · kg⁻¹ · h⁻¹) was performed. Endoscopic duodenal biopsies were taken. Leucine and phenylalanine enrichments were assessed by using gas chromatography-mass spectrometry in duodenal proteins and the intracellular free amino acids pool to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates and macroarrays, respectively. RESULTS: The FSR and proteasome activity were not different after the glutamine supply compared with after maltodextrins alone. In contrast, the FSR increased (1.7-fold increase; P < 0.05) after protein-powder delivery without modification of total proteasome activity. The protein powder increased insulinemia, PI3 kinase, and erk phosphorylation but did not affect the mammalian target of rapamycin (mTOR) pathway and mitogen-activated protein kinase signal-integrating kinase 1 phosphorylation. A trend for an increase of eukaryotic translation initiation factor 4E phosphorylation was observed (P = 0.07). CONCLUSION: In the carbohydrate fed state, enteral proteins but not glutamine increased duodenal protein synthesis through an mTOR independent pathway in humans.

Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152).

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.

Modulation of K11-linkage formation by variable loop residues within UbcH5A.

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.

Influence of leucine on protein metabolism, phosphokinase expression, and cell proliferation in human duodenum1,3.

BACKGROUND: Although leucine increases protein anabolism through the mammalian target of rapamycin (mTOR) pathway in human muscles, its effects on intestinal mucosal proteins remain unknown. OBJECTIVE: We aimed to assess the effects of leucine on duodenal protein metabolism in healthy humans and to elucidate the signaling pathways involved. DESIGN: Eleven healthy volunteers received for 5 h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g . kg(-1) . h(-1)) or maltodextrins and leucine (0.035 g . kg(-1) . h(-1)) simultaneously with a continuous intravenous infusion of [(2)H(5)]phenylalanine (9 µmol . kg(-1) .h(-1)). Endoscopic duodenal biopsy samples were collected and frozen until analyzed. Phenylalanine enrichment was assessed by gas chromatography-mass spectrometry in duodenal protein and in free intracellular amino acid pools used as precursor to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates or macroarrays, respectively. RESULTS: Leucine supplementation slightly reduced FSR (mean ± SEM: 81.3 ± 6.3%/d) compared with maltodextrins alone (91.7 ± 8.5%/d; P = 0.0537). In addition, total proteasome activity decreased significantly with leucine (236 ± 21 compared with 400 ± 58 relative fluorescence units/µg protein; P < 0.05), with no modification of chymotrypsin-like, trypsin-like, caspase-like, or peptidase activities. Leucine did not affect the mTOR pathway but did increase the phosphorylation states of PI3K, Akt, AMPK, p38 MAPK, JNK, GSK-3α/ß, STAT3, and STAT5 and increased cyclin D1 mRNA concentrations, which suggested that leucine may enhance cell proliferation. CONCLUSION: Enteral leucine supplementation decreased proteasome activity in duodenal mucosa and enhanced cell proliferation through the PI3K/Akt/GSK-3α/ß-catenin pathway. This trial was registered at clinicaltrials.gov as NCT01254110.

Antagonists induce a conformational change in cIAP1 that promotes autoubiquitination.

Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.

Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson's disease-associated protein DJ-1.

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease.

Antagonism of c-IAP and XIAP proteins is required for efficient induction of cell death by small-molecule IAP antagonists.

The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival, which makes them attractive targets for therapeutic intervention in cancers. Herein, we describe the structure-based design of IAP antagonists with high affinities and selectivity (>2000-fold) for c-IAP1 over XIAP and their functional characterization as activators of apoptosis in tumor cells. Although capable of inducing cell death and preventing clonogenic survival, c-IAP-selective antagonists are significantly less potent in promoting apoptosis when compared to pan-selective compounds. However, both pan-IAP- and c-IAP-selective antagonists stimulate c-IAP1 and c-IAP2 degradation and activation of NF-kappaB pathways with comparable potencies. Therefore, although compounds that specifically target c-IAP1 and c-IAP2 are capable of inducing apoptosis, antagonism of the c-IAP proteins and XIAP is required for efficient induction of cancer cell death by IAP antagonists.

Detection of DNA double-strand breaks using gammaH2AX after MRI exposure at 3 Tesla: an in vitro study.

PURPOSE: To evaluate the effects of the static magnetic field and typical imaging sequences of a high-field magnetic resonance scanner (3 Tesla) on the induction of double-strand breaks (DSBs) in two different human cell lines. MATERIALS AND METHODS: Human promyelocytic leukemia cells (HL-60) and human acute myeloid leukemia cells (KG-1a) were exposed to the static magnetic field alone and to turbo spin-echo (TSE) and gradient-echo (GE) sequences. Flow cytometry was used to quantify gammaH2AX (serine 139 phosphorylated form of histone H2AX) expression of antibody-stained cells as a marker for deoxyribonucleic acid (DNA) DSBs one hour and 24 hours after magnetic field exposure. X-ray-treated cells were used as positive control. RESULTS: Neither exposure to the static magnetic field alone nor to the applied imaging sequences showed significant differences in gammaH2AX expression between exposed and sham-exposed cells. X-ray-treated cells as positive control showed a significant increase in gammaH2AX expression. CONCLUSION: The static magnetic field alone and MRI sequences at 3 Tesla have no effect on the induction of DSBs in HL-60 and KG-1a cells.