Five subtypes of benign paroxysmal positional vertigo.

BACKGROUND: Benign paroxysmal positional vertigo is classified into five subtypes according to the features of positional nystagmus: lateral canalolithiasis, lateral light cupula, lateral heavy cupula, posterior canalolithiasis and posterior heavy cupula. OBJECTIVES: The first aim of the study was to clarify whether the lateral canal type or posterior canal type was more common. The second aim of the study was to assess the aetiology of benign paroxysmal positional vertigo by investigating the onset time of each subtype. METHODS: The subjects were 512 consecutive patients with benign paroxysmal positional vertigo. The patients were prospectively aggregated, and interviews were used to evaluate onset time. RESULTS: The lateral canal type (55.5 per cent) was more common than the posterior canal type (44.5 per cent). Time of awakening was the most common onset time in every subtype. CONCLUSION: The incidence of lateral canal type is higher than that of posterior canal type. The aetiology of benign paroxysmal positional vertigo is closely related to sleep.

PO-16 - ASK1 regulates tumor lung metastasis and platelet functions.

INTRODUCTION: Apoptosis signal-regulating kinase 1 (ASK1) is a MAP3K in the JNK and p38 MAPK pathways and responds to various stresses. Accumulating evidence indicates that ASK1 plays important roles in tumorigenesis by regulating apoptosis and inflammation. However, little is known about ASK1's roles in tumor metastasis. AIM: To investigate ASK1's roles in tumor metastasis. MATERIALS AND METHODS: We performed experimental lung metastasis model by intravenous injection of Lewis lung carcinoma cells constitutively expressing luciferase (3LL-Luc2 cells). As for the analysis of platelet functions, tail bleeding assay and ferric chloride-induced thrombosis model were utilized. RESULTS: We measured the transition of luciferase activity of the lung lysates up to 7 days as an indicator of lung metastasis. ASK1-/- mice showed markedly lower luciferase activity as early as 3 hours after injection compared to WT mice; hence ASK1 appears to be involved in the early stage of tumor lung metastasis, which is prior to the extravasation of tumor cells. Platelets aggregate and adhere to tumor cells in the early stage and are known to support hematogenous metastasis. ASK1-/- mice were normal in hematological parameters including platelet number, while analysis by western blot revealed that platelets of ASK1-/- mice exhibited markedly reduced phosphorylation of JNK and p38, both of which have been reported to regulate platelet functions such as platelet aggregation. We found that platelets of ASK1-/- mice were less responsive to specific aggregation agonists and that ASK1-/- mice showed bleeding tendency and defect in thrombosis. These phenotypes were also observed in megakaryocyte and platelet-specific ASK1 deficient mice. CONCLUSIONS: It is suggested that impaired platelet functions caused by ASK1 deficiency in platelets may attenuate tumor lung metastasis.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury.

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway.

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1ß. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

ASK1 deficiency attenuates neural cell death in GLAST-deficient mice, a model of normal tension glaucoma.

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced retinal ganglion cell (RGC) apoptosis. In the mammalian retina, glutamate/aspartate transporter (GLAST) is a major glutamate transporter, and the loss of GLAST leads to optic nerve degeneration similar to normal tension glaucoma (NTG). In GLAST⁻(/)⁻ mice, the glutathione level in the retina is decreased, suggesting the involvement of oxidative stress in NTG pathogenesis. To test this hypothesis, we examined the histology and visual function of GLAST(+/)⁻:ASK1⁻(/)⁻ and GLAST⁻(/)⁻:ASK1⁻(/)⁻ mice by multifocal electroretinograms. ASK1 deficiency protected RGCs and decreased the number of degenerating axons in the optic nerve. Consistent with this finding, visual function was significantly improved in GLAST(+/)⁻:ASK1⁻(/)⁻ and GLAST⁻(/)⁻:ASK1⁻(/)⁻ mice compared with GLAST(+/)⁻ and GLAST⁻(/)⁻ mice, respectively. The loss of ASK1 had no effects on the production of glutathione or malondialdehyde in the retina or on the intraocular pressure. Tumor necrosis factor (TNF)-induced activation of p38 MAPK and the production of inducible nitric oxide synthase were suppressed in ASK1-deficient Müller glial cells. In addition, TNF-induced cell death was suppressed in ASK1-deficient RGCs. These results suggest that ASK1 activation is involved in NTG-like pathology in both neural and glial cells and that interrupting ASK1-dependent pathways could be beneficial in the treatment of glaucoma, including NTG.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death.

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.

Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.

Enzymatic modification of low-density lipoprotein (LDL) as it probably occurs in the arterial intima drastically increases its cytotoxicity, which could be relevant for the progression of atherosclerotic lesions. LDL was treated with a protease and cholesterylesterase to generate a derivative similar to lesional LDL, with a high content of free cholesterol and fatty acids. Exposure of endothelial cells to the enzymatically modified lipoprotein (E-LDL), but not to native or oxidized LDL, resulted in programmed cell death. Apoptosis was triggered by apoptosis signal-regulating kinase 1 dependent phosphorylation of p38. Depletion and reconstitution experiments identified free fatty acids (FFA) as the triggers of this pathway. Levels of FFA in native LDL are low and the lipoprotein is therefore not cytotoxic; enzymatic cleavage of cholesterylesters liberates FFA that can rapidly trigger an apoptosis signaling cascade in neighboring cells. Blockade of this pathway can rescue cells from death.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H(2)O(2)). A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H(2)O(2) treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H(2)O(2)-induced sustained activation of ASK1 but also ASK1-dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1-JNK/p38 pathways by negative feedback.

Roles of the telencephalic cells and their chondroitin sulfate proteoglycans in delimiting an anterior border of the retinal pathway.

The axons of the retinal ganglion cells run on the diencephalotelencephalic boundary on their way to the tectum; however, they do not invade the telencephalon anteriorly. To investigate the mechanisms that prevent the retinal axons from entering the telencephalic territory, the effects of the telencephalic cells were examined on the outgrowth of the retinal axons in vitro; the retinal outgrowth was selectively inhibited by the cellular substrate derived from the telencephalon. The responsible factor for the selective inhibition was, furthermore, found in the telencephalic membranes and the fraction of peripheral membrane molecules from the telencephalon. Because the inhibitory effect was destroyed by chondroitinase ABC but not by heat, this inhibition was attributable to the carbohydrate chains of chondroitin sulfate proteoglycans (CSPGs) adhering to the membranes of the telencephalic cells. To understand the function of the telencephalic CSPGs on the retinal pathfinding in vivo, their carbohydrate chains [chondroitin sulfate glycosaminoglycan (CS-GAG)] were removed from the embryonic brains by intraventricular injection of chondroitinase ABC; the removal of CS-GAG resulted in an anterior enlargement of the optic tract. The results indicate that the telencephalic cells delimit the anterior border of the optic tract with their CSPGs and prevent the retinal axons from aberrantly entering the anterior territory.

ASK1 resistant neuroblastoma is deficient in activation of p38 kinase.

Apoptosis Signal-regulating Kinase 1 (ASK1) is known to either induce apoptosis or differentiation in various cell lines of neuronal origin. We analyzed the effect of the constitutively active mutant of ASK1 (ASK1-Delta N) in an adenoviral vector in four neuroblastoma cell lines, two murine, C1300 and NXS2, and two human, SH-SY5Y and IMR-32. Already after 24 h upon infection, C1300 and SH-SY5Y cells arrested in growth when judged by [(3)H]thymidine incorporation, and the majority of the cells demonstrated apoptotic appearance, which was confirmed by DNA-laddering in gel electrophoresis. In contrast, NXS2 and IMR-32 cell lines remained unaffected. Immunoblotting revealed strongly phosphorylated p38 MAPK accompanied by weakly phosphorylated JNK in C1300 and SH-SY5Y, whereas none of these kinases were activated by adenoviruses expressing the kinase negative ASK1 mutant or beta-galactosidase. There was no expression of phosphorylated kinases in IMR-32 cells, but NXS2 showed a faint band of phosphorylated p38 MAPK. Addition of the p38 MAPK specific inhibitor, SB203580, protected C1300 and SH-SY5Y cells from apoptosis induced by ASK1-Delta N. The anti-neoplastic agent, paclitaxel, activates ASK1 and JNK, and promotes the in vitro assembly of stable microtubules. Addition of 10 nM paclitaxel sensitised the NXS2 cell line to ASK1-induced cell death. Our results indicate that ASK1 induces apoptosis in neuroblastoma cells mainly via the p38 MAPK pathway, and resistant neuroblastoma cells can be sensitised to ASK1 by paclitaxel.

Tumor growth inhibition by arsenic trioxide (As2O3) in the orthotopic metastasis model of androgen-independent prostate cancer.

Arsenic trioxide (As2O3) induces clinical remission of patients with acute promyelocytic leukemia. As a novel anticancer agent for treatment of solid cancers, As2O3 is promising, but no in vivo experimental investigations of its efficacy on solid cancers have been done at clinically obtained concentrations. In addition, the cell death mechanism of As2O3 has yet to be clarified, especially in solid cancers. In this study, human androgen-independent prostate cancer cell lines, PC-3, DU-145, and TSU-PR1 were examined as cellular models for As2O3 treatment, and As2O3-induced cell death and inhibition of cell growth and colony formation were evaluated. The involvement of p38, c-Jun NH2-terminal kinase (JNK), caspase-3, and reactive oxygen species (ROS) were investigated in As2O3-induced cell death. Finally, As2O3 was administered to severe combined immunodeficient mice inoculated orthotopically with PC-3 cells to estimate in vivo efficacy. In all three of the cell lines, at high concentrations, As2O3 induced apoptosis and, at low concentrations, growth inhibition. As2O3 activated p38, JNK, and caspase-3 dose dependently. Treatment with the p38 inhibitor and over-expression of dominant-negative JNK did not guard against As2O3-induced cell death. In contrast with partial protection by the caspase-3 inhibitor, the antioxidant N-acetyl-L-cysteine gave marked protection from As2O3-induced apoptosis and eliminated the activation of p38, JNK, and caspase-3, and the generation of ROS. The orthotopic murine metastasis model showed in vivo tumor growth inhibition in orthotopic and metastatic lesions with no signs of toxicity. This study establishes that As2O3 provides a novel, safe approach for treatment of androgen-independent prostate cancer. Generation of ROS as a therapeutic target for the potentiation of As2O3-induced apoptosis also was shown.

Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1.

Coordination and balance between cell survival and apoptosis is crucial for normal development and homeostasis of multicellular organisms. Defects in control of this balance may contribute to a variety of diseases including cancer, autoimmune and neurodegenerative conditions. Although a large number of pro- and anti-apoptotic factors acting for or against the final death event have been and are being discovered at an extraordinary pace with the recent progress in this area, the molecular mechanisms determining whether a cell lives or dies are not fully understood. Phosphorylation and dephosphorylation of intracellular effector molecules are the most common and important regulatory mechanisms in signal transduction and control a variety of cellular events from cell growth to apoptosis. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase kinase kinase family, which activates both the SEK1-JNK and MKK3/6-p38 MAP kinase pathways and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. This review provides recent findings on the molecular mechanisms which determine cell fate such as survival, proliferation, differentiation or apoptosis, with special focus on the regulatory mechanisms of ASK1-mediated apoptosis.

The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1.

CDC25A phosphatase promotes cell cycle progression by activating G(1) cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts. In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening. ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses. Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm. The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A. This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1. Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli. Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression. These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.

HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell.

In vivo infection of lymphatic tissues by the human immunodeficiency virus type 1 (HIV-1) leads to enhanced apoptosis, which prominently involves uninfected bystander cells. Increased killing of such bystander cells is mediated in part through Nef induction of Fas ligand (FasL) expression on the surface of the virally infected T cells. The subsequent interaction of FasL with Fas (CD95) displayed on neighbouring cells, including HIV-1-specific cytotoxic T lymphocytes, may lead to bystander cell killing and thus forms an important mechanism of immune evasion. As HIV-1 also enhances Fas expression on virally infected cells, it is unclear how these hosts avoid rapid cell-autonomous apoptosis mediated through cis ligation of Fas by FasL. Here we show that HIV-1 Nef associates with and inhibits apoptosis signal-regulating kinase 1 (ASK1), a serine/threonine kinase that forms a common and key signalling intermediate in the Fas and tumour-necrosis factor-alpha (TNFalpha) death-signalling pathways. The interaction of Nef with ASK1 inhibits both Fas- and TNFalpha-mediated apoptosis, as well as the activation of the downstream c-Jun amino-terminal kinase. Our findings reveal a strategy by which HIV-1 Nef promotes the killing of bystander cells through the induction of FasL, while simultaneously protecting the HIV-1-infected host cell from these same pro-apoptotic signals through its interference with ASK1 function.

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.

Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H(2)O(2), and activates c-Jun NH(2)-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H(2)O(2)-induced sustained activations of JNK and p38 are lost in ASK1(-/-) embryonic fibroblasts, and that ASK1(-/-) cells are resistant to TNF- and H(2)O(2)-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1-JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.