Ultraviolet light induces redox reaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3–4%) and Ret-MEN2B (1–2%) and large percentages of Ret-MEN2A (30–40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond–mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond–mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain–deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1–C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.     

RET tyrosine kinase signaling in development and cancer

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.     

Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用

在前期研究中,已发现人瘦素（leptin）在体外再折叠过程中会形成稳定的二聚体,但其二聚化机制尚不清楚. 本研究旨在分析瘦素二聚体的结构特性,并重点研究体外再折叠过程中瘦素二聚化的机制. 相较与瘦素单体,瘦素二聚体保留了约75％免疫活性及15％受体结合活性,同时显示出明显慢的天然电泳迁移率. 圆二色性分析显示,二聚体基本保留了单体α螺旋索结构特征. 还原性及非还原性凝胶电泳分析和自由巯基测定结果表明,瘦素二聚体是由一对分子间二硫键连接2个单体而成的.为了确定瘦素二聚化过程中起主导作用的分子间二硫键,利用PCR定点突变技术构建了C96S和C146S两个突变体瘦素. 通过分析C96S及C146S突变体瘦素的体外再折叠特性及过程,并与野生型瘦素相比较,揭示C96S瘦素的二聚体显示出与野生型瘦素二聚体相似的特性,而C146S瘦素不能形成结构稳定的二聚体. 以上研究结果表明,Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用.     

The dimerization of PSGL‐1 is driven by the transmembrane domain via a leucine zipper motif

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is a homodimeric mucin ligand that is important to mediate the earliest adhesive event during an inflammatory response by rapidly forming and dissociating the selectin‐ligand adhesive bonds. Recent research indicates that the noncovalent associations between the PSGL‐1 transmembrane domains (TMDs) can substitute for the C320‐dependent covalent bond to mediate the dimerization of PSGL‐1. In this article, we combined TOXCAT assays and molecular dynamics (MD) simulations to probe the mechanism of PSGL‐1 dimerization. The results of TOXCAT assays and Martini coarse‐grained molecular dynamics (CG MD) simulations demonstrated that PSGL‐1 TMDs strongly dimerized in a natural membrane and a leucine zipper motif was responsible for the noncovalent dimerization of PSGL‐1 TMD since mutations of the residues that occupied a or d positions in an (abcdefg)n leucine heptad repeat motif significantly reduced the dimer activity. Furthermore, we studied the effects of the disulfide bond on the PSGL‐1 dimer using MD simulations. The disulfide bond was critical to form the leucine zipper structure, by which the disulfide bond further improved the stability of the PSGL‐1 dimer. These findings provide insights to understand the transmembrane association of PSGL‐1 that is an important structural basis for PSGL‐1 preferentially binding to P‐selectin to achieve its biochemical and biophysical functions.     

Inducible dimerization of RET reveals a specific AKT deregulation in oncogenic signaling

Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation.     

Sorafenib functions to potently suppress RET tyrosine kinase activity by direct enzymatic inhibition and promoting RET lysosomal degradation independent of proteasomal targeting

Germ line missense mutations in the RET (rearranged during transfection) oncogene are the cause of multiple endocrine neoplasia, type 2 (MEN2), but at present surgery is the only treatment available for MEN2 patients. In this study, the ability of Sorafenib (BAY 43-9006) to act as a RET inhibitor was investigated. Sorafenib inhibited the activity of purified recombinant kinase domain of wild type RET and RET(V804M) with IC(50) values of 5.9 and 7.9 nm, respectively. Interestingly, these values were 6-7-fold lower than the IC(50) for the inhibition of B-RAF(V600E). In cell-based assays, Sorafenib inhibited the kinase activity and signaling of wild type and oncogenic RET in MEN2 tumor and established cell lines at a concentration between 15 and 150 nm. In contrast, inhibition of oncogenic B-RAF- or epidermal growth factor-induced ERK1/2 phosphorylation required micromolar concentrations of Sorafenib demonstrating the high specificity of this drug in targeting RET. Moreover, prolonged exposure to Sorafenib resulted in inhibition of cell proliferation and RET protein degradation. Using lysosomal and proteasomal inhibitors, we demonstrate that Sorafenib induces RET lysosomal degradation independent of proteasomal targeting. Furthermore, we provide a structural model of the Sorafenib.RET complex in which Sorafenib binds to and induces the DFG(out) conformation of the RET kinase domain. These results strengthen the argument that Sorafenib may be effective in the treatment of MEN2 patients. In addition, because inhibition of RET is not impaired by mutation of the Val(804) gatekeeper residue, MEN2 tumors may be less susceptible to acquired Sorafenib resistance.     

Regulatory elements and functional implication for the formation of dimeric visinin‐like protein‐1

Size exclusion chromatographic analyses showed that Ca²⁺‐free VILIP‐1 contained both monomeric and dimeric forms, while no appreciable dimerization was noted with Ca²⁺‐free VILIP‐3. Swapping of EF‐hands 3 and 4 of VILIP‐1 with those of VILIP‐3 caused the inability of the resulting chimeric protein to form dimeric protein. Nonreducing SDS‐PAGE analyses revealed that most of the dimeric VILIP‐1 was noncovalently bound together. Reduced glutathione (GSH)/oxidized glutathione (GSSG) treatment notably enhanced the formation of disulfide‐linked VILIP‐1 dimer, while Ca²⁺ and Mg²⁺ enhanced disulfide dimerization of VILIP‐1 marginally in the presence of thiol compounds. Cys‐187 at the C‐terminus of VILIP‐1 contributed greatly to form S‐S‐crosslinked dimer as revealed by mutagenesis studies. The ability of GSH/GSSG‐treated VILIP‐1 to activate guanylyl cyclase B was reduced by substituting Cys‐187 with Ala. Together with disulfide dimer of VILIP‐1 detected in rat brain extracts, our data may imply the functional contribution of disulfide dimer to the interaction of VILIP‐1 with its physiological target(s). Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro folding of methionine‐arginine human lyspro‐proinsulin S‐sulfonate—Disulfide formation pathways and factors controlling yield

We investigated the in vitro folding of an oxidized proinsulin (methionine‐arginine human lyspro‐proinsulin S‐sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5–11). Folding intermediates were detected and characterized by means of matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS), reversed‐phase chromatography (RPC), size‐exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI‐MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI‐MS analysis of Glu‐C digested, purified intermediates indicated that an intra‐A‐chain disulfide bond formed first among A6, A7, and A11. Various non‐native intra‐A (A20 with A6, A7, or A11), intra‐B (between B7 and B19), and inter‐A‐B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non‐native intra‐A and intra‐B bonds. At a cysteine‐to‐proinsulin‐SH ratio of 3.5, all intermediates with the non‐native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine‐to‐proinsulin‐SH ratio, some intermediates and folded MR‐KPB‐hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine‐to‐protein ratio, can improve folding yield significantly. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin

The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.     

Examination of the structure/function relationship in the exchangeable apolipoprotein,apolipophorin‐III

Exchangeable apolipoproteins are proteins that reversibly bind lipoprotein particles to facilitate their transport in vivo. The structure/function relationship of apolipophorin‐III (apo‐III), the only insect exchangeable apolipoprotein, has been investigated by examining the association of this protein with lipid vesicles. The importance of a conserved leucine residue, reported to be essential for apo‐III binding to lipids, has been evaluated through site‐directed mutagenesis. A unique cysteine replaces the conserved leucine at position 30 in recombinant apo‐III (L30C protein). This substitution results in the covalent dimerization of the apo‐III mutant via a disulfide bond. The cysteine mutation causes no difference in surface hydrophobicity of the L30C proteins when compared to the wild type apo‐III. Wild type apo‐III, L30C monomer, and L30C dimer associate with dimyristoylphosphatidylcholine (DMAC) vesicles in a similar manner, resulting in a reduction of turbidity of a phospholipid vesicle suspension. Analysis with transmission electron microscopy (TEM) reveals disk‐like complexes identical to those previously reported with the wild type apo‐III. Because the mutation of the conserved leucine seems to affect the solution behavior and surface hydrophobicity of apo‐III, this residue is likely to be exposed to the aqueous environment. However, the similar behaviors of the wild type protein, the L30C monomer, and L30C dimer with respect to the binding of phospholipid vesicles suggest that this residue is not absolutely required for the protein binding to hydrophobic or amphiphilic interfaces. © 1999 John Wiley & Sons, Inc. Biopoly 50: 486–495, 1999     

MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

Redesigning the procaspase‐8 dimer interface for improved dimerization

Caspase‐8 is a cysteine directed aspartate‐specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase‐8 depends on adaptor‐induced dimerization of procaspase‐8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase‐8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase‐3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase‐8 is a monomer while procaspase‐3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase‐8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase‐8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase‐8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase‐8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.     

LncRNA‐MIF,a c‐Myc‐activated long non‐coding RNA,suppresses glycolysis by promoting Fbxw7‐mediated c‐Myc degradation

The c‐Myc proto‐oncogene is activated in more than half of all human cancers. However, the precise regulation of c‐Myc protein stability is unknown. Here, we show that the lncRNA‐MIF (c‐Myc inhibitory factor), a c‐Myc‐induced long non‐coding RNA, is a competing endogenous RNA for miR‐586 and attenuates the inhibitory effect of miR‐586 on Fbxw7, an E3 ligase for c‐Myc, leading to increased Fbxw7 expression and subsequent c‐Myc degradation. Our data reveal the existence of a feedback loop between c‐Myc and lncRNA‐MIF, through which c‐Myc protein stability is finely controlled. Additionally, we show that the lncRNA‐MIF inhibits aerobic glycolysis and tumorigenesis by suppressing c‐Myc and miR‐586.     

Effects of siRNA on RET/PTC3 Junction Oncogene in Papillary Thyroid Carcinoma: From Molecular and Cellular Studies to Preclinical Investigations

RET/PTC3 junction oncogene is typical of radiation-induced childhood papillary thyroid carcinoma (PTC) with a short latency period. Since, RET/PTC3 is only present in the tumour cells, thus represents an interesting target for specific therapy by small interfering RNA (siRNA). Our aim is to demonstrate in vitro and in vivo molecular and cellular effects of siRNA on RET/PTC3 knockdown for therapeutic application.First, we established a novel cell line stably expressing RET/PTC3 junction oncogene, named RP3 which was found tumorigenic in nude mice compared to NIH/3T3 mouse fibroblasts. Among four siRNAs and five concentrations tested against RET/PTC3, an efficient siRNA RET/PTC3 and an appropriate dose (50 nM) were selected which showed significant inhibition (p<0.001) of gene (RT-qPCR) and protein (Western blot) expressions. This siRNA was found efficient in RP3 cells (harbouring RET/PTC3) but non-efficient in BHP10-3 SCmice cell line (harbouring RET/PTC1) showing that a specific siRNA against fusion sequence is required to target the junction oncogene. In vitro siRNA RET/PTC3 showed significant (p<0.001) inhibitory effects on RP3 cell viability (MTT assay) and on invasion/migration (IncuCyte scratch test) with blockage of cell cycle at G0/G1 phase (flow cytometry) and induced apoptosis by caspase-3 and PARP1 cleavage (WB). After intravenous injection in nude mice, respective squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC3 significantly (p<0.001) reduced RP3 tumour growth, oncogene and oncoprotein expressions, induced apoptosis and partially restored differentiation (decrease in Ki67). Hence, our findings highly support the use of siRNA RET/PTC3-SQ NPs as a new promising treatment for patients affected by PTC expressing RET/PTC3.     

A two-hit model for development of multiple endocrine neoplasia type 2B by RET mutations

Multiple endocrine neoplasia (MEN) type 2B mutations have been reported at methionine 918 or alanine 883 in the tyrosine kinase domain of the RET proto-oncogene. Recently, a new combination of two germline missense mutations at valine 804 and tyrosine 806 was identified in a patient with MEN 2B-like clinical phenotypes including medullary thyroid carcinoma, mucosal neuroma, and marfanoid habitus. In this case, valine 804 and tyrosine 806 were replaced with methionine and cysteine, respectively. In the present study, biological activities of RET with these new mutations were compared with those with known MEN 2A or MEN 2B mutations. The transforming activity of RET with the V804M/Y806C mutation was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. Like RET with the M918T or A883F MEN 2B mutation, the transforming activity of RET with the V804M/Y806C mutation was not affected by substitution of phenylalanine for tyrosine 905 that abolished the activity of RET with the MEN 2A mutation. On the other hand, substitution of phenylalanine for tyrosines 864 and 952 drastically diminished the activity of RET with the V804M/Y806C, M918T or A883F mutation, suggesting that these three mutant proteins have similar biological properties.     

Differential effects of leukocyte common antigen-related protein on biochemical and biological activities of RET-MEN2A and RET-MEN2B mutant proteins

Protein-tyrosine-phosphatases (PTPs), in conjunction with protein-tyrosine kinases, play essential regulatory roles in diverse cellular activities by modulating the phosphorylation state of target proteins. Leukocyte common antigen-related (LAR) protein is a widely expressed receptor-type protein-tyrosine-phosphatase that is implicated in the regulation of intracellular signaling triggered by both cell adhesion and peptide growth factors. The gene for LAR is localized to human chromosome 1p32, a region frequently deleted in tumors of neuroectodermal origin, including neuroblastoma, pheochromocytoma, and medullary thyroid carcinoma. On the other hand, the RET gene codes for a transmembrane tyrosine kinase and is responsible for the development of multiple endocrine neoplasia (MEN) 2A and 2B. To explore the potential role of LAR in RET tyrosine kinase activity and RET-induced signal transduction, we cotransfected LAR and RET with a MEN2A or MEN2B mutation (designated RET-MEN2A or RET-MEN2B) into the NIH 3T3 cell line. Here we show that LAR reduces the constitutive tyrosine autophosphorylation and kinase activity of RET-MEN2A but not RET-MEN2B, accompanying a significant decrease of phosphorylation of phospholipase Cgamma, AKT, and ERK1/2. Interestingly, LAR expression significantly decreased the levels of disulfide-linked RET-MEN2A dimerization. Moreover, reduced oncogenic activity of RET-MEN2A by overexpression of LAR was observed both by an in vitro colony formation assay and by in vivo tumorigenicity in scid mice. These results thus suggest that LAR may contribute to deactivation of the RET-MEN2A mutant protein and reduction of its oncogenic activity in vivo.     

Differential interaction of Enigma protein with the two RET isoforms

The receptor tyrosine kinase RET, with a known role in embryonic development and in human pathologies, is alternatively spliced to yield at least two functional isoforms, which differ only in their carboxyl terminal. Enigma protein is a member of the PDZ-LIM family and is known to interact with the short isoform of RET/PTC2, a chimeric oncoprotein isolated from papillary thyroid carcinoma. Here, we show that Enigma also interacts in intact cells with the short isoform of RET-wt and of its pathologic mutants associated to MEN2 syndromes, RET-C634R and RET-M918T. In contrast, Enigma binds all the corresponding RET long isoforms very poorly and colocalizes with short but not long RET/PTC2 isoforms. The RET docking tyrosine for Enigma is the last but one before the divergence between the two isoforms and we demonstrated that short-isoform-specific amino acid residues +2 to +4 to this tyrosine are required for the interaction of RET/PTC2 with Enigma.     

Arsenate‐induced apoptosis in murine embryonic maxillary mesenchymal cells via mitochondrial‐mediated oxidative injury

BACKGROUND : Arsenic is a ubiquitous element that is a potential carcinogen and teratogen and can cause adverse developmental outcomes. Arsenic exerts its toxic effects through the generation of reactive oxygen species (ROS) that include hydrogen peroxide (H₂O₂), superoxide‐derived hydroxyl ion, and peroxyl radicals. However, the molecular mechanisms by which arsenic induces cytotoxicity in murine embryonic maxillary mesenchymal (MEMM) cells are undefined. METHODS : MEMM cells in culture were treated with different concentrations of pentavalent sodium arsenate [As (V)] for 24 or 48 hr and various end points measured. RESULTS : Treatment of MEMM cells with the pentavalent form of inorganic arsenic resulted in caspase‐mediated apoptosis, accompanied by generation of ROS and disruption of mitochondrial membrane potential. Treatment with caspase inhibitors markedly blocked apoptosis. In addition, the free radical scavenger N‐acetylcysteine dramatically attenuated arsenic‐mediated ROS production and apoptosis, and exposure to arsenate increased Bax and decreased Bcl protein levels in MEMM cells. CONCLUSIONS : Taken together, these findings suggest that in MEMM cells arsenate‐mediated oxidative injury acts as an early and upstream initiator of the cell death cascade, triggering cytotoxicity, mitochondrial dysfunction, altered Bcl/Bax protein ratios, and activation of caspase‐9. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.