Clinical profiling of MRD48 and functional characterization of two novel pathogenic RAC1 variants.

RAC1 is a member of the Rac/Rho GTPase subfamily within the RAS superfamily of small GTP-binding proteins, comprising 3 paralogs playing a critical role in actin cytoskeleton remodeling, cell migration, proliferation and differentiation. De novo missense variants in RAC1 are associated with a rare neurodevelopmental disorder (MRD48) characterized by DD/ID and brain abnormalities coupled with a wide range of additional features. Structural and functional studies have documented either a dominant negative or constitutively active behavior for a subset of mutations. Here, we describe two individuals with previously unreported de novo missense RAC1 variants. We functionally demonstrate their pathogenicity proving a gain-of-function (GoF) effect for both. By reviewing the clinical features of these two individuals and the previously published MRD48 subjects, we further delineate the clinical profile of the disorder, confirming its phenotypic variability. Moreover, we compare the main features of MRD48 with the neurodevelopmental disease caused by GoF variants in the paralog RAC3, highlighting similarities and differences. Finally, we review all previously reported variants in RAC proteins and in the closely related CDC42, providing an updated overview of the spectrum and hotspots of pathogenic variants affecting these functionally related GTPases.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish.

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.

Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis.

We have recently provided new evidence for a role of p75NTR receptor and its preferential ligand proNGF in amplifying inflammatory responses in synovial mononuclear cells of chronic arthritis patients. In the present study, to better investigate how activation of the p75NTR/proNGF axis impacts synovial inflammation, we have studied the effects of proNGF on fibroblast-like synoviocytes (FLS), which play a central role in modulating local immune responses and in activating pro-inflammatory pathways. Using single cell RNA sequencing in synovial tissues from active and treatment-naïve rheumatoid arthritis (RA) patients, we demonstrated that p75NTR and sortilin, which form a high affinity receptor complex for proNGF, are highly expressed in PRG4pos lining and THY1posCOL1A1pos sublining fibroblast clusters in RA synovia but decreased in RA patients in sustained clinical remission. In ex vivo experiments we found that FLS from rheumatoid arthritis patients (RA-FLS) retained in vitro a markedly higher expression of p75NTR and sortilin than FLS from osteoarthritis patients (OA-FLS). Inflammatory stimuli further up-regulated p75NTR expression and induced endogenous production of proNGF in RA-FLS, leading to an autocrine activation of the proNGF/p75NTR pathway that results in an increased release of pro-inflammatory cytokines. Our data on the inhibition of p75NTR receptor, which reduced the release of IL-1ß, IL-6 and TNF-α, further confirmed the key role of p75NTR activation in regulating inflammatory cytokine production. In a set of ex vivo experiments, we used RA-FLS and cultured them in the presence of synovial fluids obtained from arthritis patients that, as we demonstrated, are characterized by a high concentration of proNGF. Our data show that the high levels of proNGF present in inflamed synovial fluids induced pro-inflammatory cytokine production by RA-FLS. The blocking of NGF binding to p75NTR using specific inhibitors led instead to the disruption of this pro-inflammatory loop, reducing activation of the p38 and JNK intracellular pathways and decreasing inflammatory cytokine production. Overall, our data demonstrate that an active proNGF/p75NTR axis promotes pro-inflammatory responses in synovial fibroblasts, thereby contributing to chronic synovial inflammation, and point to the possible use of p75NTR inhibitors as a novel therapeutic approach in chronic arthritis.

Expanding the molecular spectrum of pathogenic SHOC2 variants underlying Mazzanti syndrome.

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.

Mutations at the C-terminus of CDC42 cause distinct hematopoietic and autoinflammatory disorders.

BACKGROUND: Pathogenic missense variants in cell division control protein 42 (CDC42) differentially affect protein function, causing a clinically wide phenotypic spectrum variably affecting neurodevelopment, hematopoiesis, and immune response. More recently, 3 variants at the C-terminus of CDC42 were proposed to similarly impact protein function and cause a novel autoinflammatory disorder. OBJECTIVES: We sought to clinically and functionally classify these variants to improve patient management. METHODS: Comparative analysis of the available clinical data and medical history of patients was performed. In vitro and in vivo studies were carried out to functionally characterize individual variants. RESULTS: Differently from what had previously been observed for the p.R186C change causing neonatal-onset cytopenia, autoinflammation, and recurrent hemophagocytic lymphohistiocytosis, p.C188Y and p.∗192Cext∗24 promoted accelerated protein degradation. Unprenylated CDC42C188Y did not behave as a membrane-bound protein, whereas the residual CDC42∗192Cext∗24 mutant replicated the CDC42R186C behavior, being targeted to the Golgi apparatus in a palmitoylation-dependent manner. Assessment of in vitro polarized migration and development in Caenorhabditis elegans documented a loss-of-function behavior of the p.C188Y and p.∗192Cext∗24 variants. Consistently, the 3 pathogenic variants were associated with different clinical presentations, with dysmorphisms, severity, and age of onset of cytopenia and extent of autoinflammation representing major differences. CONCLUSIONS: Pathogenic variants at the CDC42 C-terminus differently impact protein stability, localization, and function, and cause different diseases, with p.R186C specifically associated with neonatal-onset pancytopenia and severe autoinflammation/hemophagocytic lymphohistiocytosis requiring emapalumab and bone marrow transplantation, and p.C188Y and p.∗192Cext∗24 causing anakinra-sensitive autoinflammation.

Activating MRAS mutations cause Noonan syndrome associated with hypertrophic cardiomyopathy.

The RASopathies are a group of genetic syndromes caused by upregulated RAS signaling. Noonan syndrome (NS), the most common entity among the RASopathies, is characterized mainly by short stature, cardiac anomalies and distinctive facial features. Mutations in multiple RAS-MAPK pathway-related genes have been associated with NS and related phenotypes. We describe two unrelated patients presenting with hypertrophic cardiomyopathy (HCM) and dysmorphic features suggestive of NS. One of them died in the neonatal period because of cardiac failure. Targeted sequencing revealed de novo MRAS variants, c.203C > T (p.Thr68Ile) and c.67G > C (p.Gly23Arg) as causative events. MRAS has only recently been related to NS based on the observation of two unrelated affected individuals with de novo variants involving the same codons here found mutated. Gly23 and Thr68 are highly conserved residues, and the corresponding codons are known hotspots for RASopathy-associated mutations in other RAS proteins. Functional analyses documented high level of activation of MRAS mutants due to impaired GTPase activity, which was associated with constitutive plasma membrane targeting, prolonged localization in non-raft microdomains, enhanced binding to PPP1CB and SHOC2 protein, and variably increased MAPK and PI3K-AKT activation. This report provides additional evidence that a narrow spectrum of activating mutations in MRAS represents another rare cause of NS, and that MRAS has to be counted among the RASopathy genes predisposing to HCM. Moreover, our findings further emphasize the relevance of the MRAS-SHOC2-PPP1CB axis in the control of MAPK signaling, and the contribution of both MAPK and PI3K-AKT pathways in MRAS functional upregulation.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

Dominant Noonan syndrome-causing LZTR1 mutations specifically affect the Kelch domain substrate-recognition surface and enhance RAS-MAPK signaling.

Noonan syndrome (NS), the most common RASopathy, is caused by mutations affecting signaling through RAS and the MAPK cascade. Recently, genome scanning has discovered novel genes implicated in NS, whose function in RAS-MAPK signaling remains obscure, suggesting the existence of unrecognized circuits contributing to signal modulation in this pathway. Among these genes, leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a functionally poorly characterized member of the BTB/POZ protein superfamily. Two classes of germline LZTR1 mutations underlie dominant and recessive forms of NS, while constitutional monoallelic, mostly inactivating, mutations in the same gene cause schwannomatosis, a cancer-prone disorder clinically distinct from NS. Here we show that dominant NS-causing LZTR1 mutations do not affect significantly protein stability and subcellular localization. We provide the first evidence that these mutations, but not the missense changes occurring as biallelic mutations in recessive NS, enhance stimulus-dependent RAS-MAPK signaling, which is triggered, at least in part, by an increased RAS protein pool. Moreover, we document that dominant NS-causing mutations do not perturb binding of LZTR1 to CUL3, a scaffold coordinating the assembly of a multimeric complex catalyzing protein ubiquitination but are predicted to affect the surface of the Kelch domain mediating substrate binding to the complex. Collectively, our data suggest a model in which LZTR1 contributes to the ubiquitinationof protein(s) functioning as positive modulator(s) of the RAS-MAPK signaling pathway. In this model, LZTR1 mutations are predicted to variably impair binding of these substrates to the multi-component ligase complex and their efficient ubiquitination and degradation, resulting in MAPK signaling upregulation.

Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.

SHOC2 subcellular shuttling requires the KEKE motif-rich region and N-terminal leucine-rich repeat domain and impacts on ERK signalling.

SHOC2 is a scaffold protein composed almost entirely by leucine-rich repeats (LRRs) and having an N-terminal region enriched in alternating lysine and glutamate/aspartate residues (KEKE motifs). SHOC2 acts as a positive modulator of the RAS-RAF-MEK-ERK signalling cascade by favouring stable RAF1 interaction with RAS. We previously reported that the p.Ser2Gly substitution in SHOC2 underlies Mazzanti syndrome, a RASopathy clinically overlapping Noonan syndrome, promoting N-myristoylation and constitutive targeting of the mutant to the plasma membrane. We also documented transient nuclear translocation of wild-type SHOC2 upon EGF stimulation, suggesting a more complex function in signal transduction.Here, we characterized the domains controlling SHOC2 shuttling between the nucleus and cytoplasm, and those contributing to SHOC2S2G mistargeting to the plasma membrane, analysed the structural organization of SHOC2's LRR motifs, and determined the impact of SHOC2 mislocalization on ERK signalling. We show that LRRs 1 to 13 constitute a structurally recognizable domain required for SHOC2 import into the nucleus and constitutive targeting of SHOC2S2G to the plasma membrane, while the KEKE motif-rich region is necessary to achieve efficient SHOC2 export from the nucleus. We also document that SHOC2S2G localizes both in raft and non-raft domains, and that it translocates to the non-raft domains following stimulation. Finally, we demonstrate that SHOC2 trapping at different subcellular sites has a diverse impact on ERK signalling strength and dynamics, suggesting a dual counteracting modulatory role of SHOC2 in the control of ERK signalling exerted at different intracellular compartments.

A mutation in PAK3 with a dual molecular effect deregulates the RAS/MAPK pathway and drives an X-linked syndromic phenotype.

Loss-of-function mutations in PAK3 contribute to non-syndromic X-linked intellectual disability (NS-XLID) by affecting dendritic spine density and morphology. Linkage analysis in a three-generation family with affected males showing ID, agenesis of corpus callosum, cerebellar hypoplasia, microcephaly and ichthyosis, revealed a candidate disease locus in Xq21.33q24 encompassing over 280 genes. Subsequent to sequencing all coding exons of the X chromosome, we identified a single novel variant within the linkage region, affecting a conserved codon of PAK3. Biochemical studies showed that, similar to previous NS-XLID-associated lesions, the predicted amino acid substitution (Lys389Asn) abolished the kinase activity of PAK3. In addition, the introduced residue conferred a dominant-negative function to the protein that drives the syndromic phenotype. Using a combination of in vitro and in vivo studies in zebrafish embryos, we show that PAK3(N389) escapes its physiologic degradation and is able to perturb MAPK signaling via an uncontrolled kinase-independent function, which in turn leads to alterations of cerebral and craniofacial structures in vivo. Our data expand the spectrum of phenotypes associated with PAK3 mutations, characterize a novel mechanism resulting in a dual molecular effect of the same mutation with a complex PAK3 functional deregulation and provide evidence for a direct functional impact of aberrant PAK3 function on MAPK signaling.

Autocrine role of angiopoietins during megakaryocytic differentiation.

The tyrosine kinase Tie-2 and its ligands Angiopoietins (Angs) transduce critical signals for angiogenesis in endothelial cells. This receptor and Ang-1 are coexpressed in hematopoietic stem cells and in a subset of megakaryocytes, though a possible role of angiopoietins in megakaryocytic differentiation/proliferation remains to be demonstrated. To investigate a possible effect of Ang-1/Ang-2 on megakaryocytic proliferation/differentiation we have used both normal CD34(+) cells induced to megakaryocytic differentiation and the UT7 cells engineered to express the thrombopoietin receptor (TPOR, also known as c-mpl, UT7/mpl). Our results indicate that Ang-1/Ang-2 may have a role in megakaryopoiesis. Particularly, Ang-2 is predominantly produced and released by immature normal megakaryocytic cells and by undifferentiated UT7/mpl cells and slightly stimulated TPO-induced cell proliferation. Ang-1 production is markedly induced during megakaryocytic differentiation/maturation and potentiated TPO-driven megakaryocytic differentiation. Blocking endogenously released angiopoietins partially inhibited megakaryocytic differentiation, particularly for that concerns the process of polyploidization. According to these data it is suggested that an autocrine angiopoietin/Tie-2 loop controls megakaryocytic proliferation and differentiation.

Multiple mechanisms for hydrogen peroxide-induced apoptosis.

The mechanisms of cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet well clarified. Here, we show that during recovery after H(2)O(2) treatment, apoptosis occurs in two different waves, peaking at 8 h (early) and 18 h (late) of recovery from oxidative stress. The two peaks are differentially modulated by a set of inhibitors of metabolic processes, which suggests that the first peak depends on DNA break formation, whereas the second may be correlated with H(2)O(2)-induced mitochondrial alterations.

Colocalization of the VEGF-R2 and the common IL-3/GM-CSF receptor beta chain to lipid rafts leads to enhanced p38 activation.

Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF-R2 and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) membrane localization and cell signalling in TF1-KDR cells (TF1 leukaemic cells that overexpress VEGF-R2/KDR). Activation of either GM-CSFR or VEGF-R2 was shown to determine the migration of both receptor elements (VEGF-R2 and the common beta-chain of the GM-CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM-CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF-R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the beta-chain of the GM-CSFR. Co-stimulation of TF1-KDR cells with both GM-CSF and VEGF-A resulted in massive migration of both the common GM-CSFR beta-chain and VEGF-R2 to lipid rafts and sustained p38 mitogen-activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM-CSF and VEGF-A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF- and GM-CSF-dependent proliferation of TF1-KDR and the survival of primary acute myeloid leukaemia blasts.

Sequential phases of Ca2+ alterations in pre-apoptotic cells.

The very early events of the intrinsic, damage-induced apoptotic pathway, i.e., upstream to Bax activation, probably consist of physico-chemical alterations (i.e., redox, pH or Ca2+ changes) rather then subtle molecular interactions, and in spite of many studies they remain unclear. One problem is that cells undergo apoptosis in an asynchronous way, leading to heterogeneity in the cell population that impairs the results of bulk analyses. In this study, we present a flow cytometric approach for studying Ca2+ alteration in apoptosis at the single cell level. By means of a multiparametric analysis, we could discriminate different sub-populations, i.e., viable and apoptotic cells and cells in secondary necrosis, and separately analyse static as well as dynamic Ca2+ parameters in each sub-population. With this approach, we have identified a set of sequential Ca2+ changes; two very early ones occur prior to any other apoptotic alterations, whereas a later change coincides with the appearance of apoptosis. Interestingly, the two pre-apoptotic changes occur simultaneously in all treated cells, i.e., at fixed times post-treatment, whereas the later one occurs at varying times, i.e., within a wide time range, concomitantly with the other apoptotic events.

Redox modulation of the apoptogenic activity of thapsigargin.

Thapsigargin (THG), a selective inhibitor of endoplasmic reticulum (ER) Ca2+-ATPases, causes the rapid emptying of ER Ca2+; in some cell types, this is accompanied by apoptosis, whereas other cells maintain viability. In order to understand the molecular determinants of such a different behavior, we explored the role of oxygen versus nitrogen radicals, by analyzing the apoptogenic ability of THG in the presence of inhibitors of glutathione or nitric oxide (NO) synthesis, respectively. We observed that oxygen radicals play a sensitizing role whereas nitrogen radicals prevent THG-dependent apoptosis, showing that the apoptogenic effect of THG is redox sensitive.

Glutathione depletion up-regulates Bcl-2 in BSO-resistant cells.

Glutathione depletion by inhibition of its synthesis with buthionine sulfoximine (BSO) is a focus of the current research in antitumor therapy, BSO being used as chemosensitizer. We had previously shown that two human tumor cell lines (U937 and HepG2) survive to treatment with BSO: BSO can elicit an apoptotic response, but the apoptotic process is aborted after cytochrome c release and before caspase activation, suggesting the development of an adaptive response (FASEB J., 1999, 13, 2031-2036). Here, we investigate the mechanisms of such an adaptation. We found that following BSO, U937 up-regulate Bcl-2 mRNA and protein levels, by a mechanism possibly involving NF-kappaB transcription factor; the increase in protein level is limited by a rapid decay of Bcl-2 in BSO-treated cells, suggesting that redox imbalance speeds up Bcl-2 turnover. BSO-dependent Bcl-2 up-regulation is associated with the ability to survive to BSO. Indeed, 1) its abrogation by CAPE or protein synthesis inhibition sensitizes U937 to BSO; 2) in a panel of four tumor lines, BSO-resistant (U937, HepG2, and HGB1) but not BSO-sensitive (BL41) cells can up-regulate Bcl-2 following GSH depletion; remarkably, only the latter are chemosensitized by BSO.

Heart infarct in NOD-SCID mice: therapeutic vasculogenesis by transplantation of human CD34+ cells and low dose CD34+KDR+ cells.

Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast: specifically, the human cord blood (CB) CD34+KDR+ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. Particularly, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in the immunocompromised NOD-SCID mouse model, and monitored the effects of myocardial transplantation of human CB CD34+ cells on cardiac function. Specifically, we compared the therapeutic effect of unseparated CD34+ cells vs. PBS and mononuclear cells (MNCs); moreover, we compared the action of the CD34+KDR+ cell subfraction vs. the CD34+KDR- subset. CD34+ cells significantly improve cardiac function after MI, as compared with PBS/MNCs. Similar beneficial actions were obtained using a 2-log lower number of CD34+KDR+ cells, while the same number of CD34+KDR- cells did not have any effects. The beneficial effect of CD34+KDR+ cells may mostly be ascribed to their notable resistance to apoptosis and to their angiogenic action, since cardiomyogenesis was limited. Altogether, our results indicate that, within the CD34+ cell population, the CD34+KDR+ fraction is responsible for the improvement in cardiac hemodynamics and hence represents the candidate active CD34+ cell subset.

GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes.