Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis

Triggering tumor necrosis factor receptor-1 (TNFR1) induces apoptosis in various cell lines. In contrast, stimulation of TNFR1 in L929sA leads to necrosis. Inhibition of HSP90, a chaperone for many kinases, by geldanamycin or radicicol shifted the response of L929sA cells to TNF from necrosis to apoptosis. This shift was blocked by CrmA but not by BCL-2 overexpression, suggesting that it occurred through activation of procaspase-8. Geldanamycin pretreatment led to a proteasome-dependent decrease in the levels of several TNFR1-interacting proteins including the kinases receptor-interacting protein, inhibitor of kappa B kinase-alpha, inhibitor of kappa B kinase-beta, and to a lesser extent the adaptors NF-kappaB essential modulator and tumor necrosis factor receptor-associated factor 2. As a consequence, NF-kappa B, p38MAPK, and JNK activation were abolished. No significant decrease in the levels of mitogen-activated protein kinases, adaptor proteins TNFR-associated death domain and Fas-associated death domain, or caspase-3, -8, and -9 could be detected. These results suggest that HSP90 client proteins play a crucial role in necrotic signaling. We conclude that inhibition of HSP90 may alter the composition of the TNFR1 complex, favoring the caspase-8-dependent apoptotic pathway. In the absence of geldanamycin, certain HSP90 client proteins may be preferentially recruited to the TNFR1 complex, promoting necrosis. Thus, the availability of proteins such as receptor-interacting protein, Fas-associated death domain, and caspase-8 can determine whether TNFR1 activation will lead to apoptosis or to necrosis.     

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.     

Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.     

Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.     

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.     

Expression and function of tumour necrosis factor-alpha-converting enzyme in the central nervous system

Tumour necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE/ADAM17) is a membrane protein belonging to the ADAM (a disintegrin and a metalloprotease) family able to cleave various membrane proteins, including the transmembrane form of TNF-alpha at its physiological processing site. Being an ADAM, TACE may mediate not only proteolysis but also adhesive interactions; however, the role of the disintegrin domain of TACE has not been studied. In the central nervous system (CNS), little is known about the physiological role of TACE, but some important pathophysiological functions have been reported recently, with both neurotoxic and neuroprotective repercussions. This article discusses and reviews the main contributions to this field of investigation addressing the expression and function of TACE in the CNS.     

Cathepsin B mediates tumor necrosis factor-induced arachidonic acid release in tumor cells

Arachidonic acid (AA) generated by cytosolic phospholipase A2 (cPLA2) has been suggested to function as a second messenger in tumor necrosis factor (TNF)-induced death signaling. Here, we show that cathepsin B-like proteases are required for the TNF-induced AA release in transformed cells. Pharmaceutical inhibitors of cathepsin B blocked TNF-induced AA release in human breast (MCF-7S1) and cervix (ME-180as) carcinoma as well as murine fibrosarcoma (WEHI-S) cells. Furthermore, TNF-induced AA release was significantly reduced in cathepsin B-deficient immortalized murine embryonic fibroblasts. Employing cPLA2-deficient MCF-7S1 cells expressing ectopic cPLA2 or cPLA2-deficient immortalized murine embryonic fibroblasts, we showed that cPLA2 is dispensable for TNF-induced AA release and death in these cells. Furthermore, TNF-induced cathepsin B-dependent AA release could be dissociated from the cathepsin B-independent cell death in MCF-7S1 cells, whereas both events required cathepsin B activity in other cell lines tested. These data suggest that cathepsin B inhibitors may prove useful not only in the direct control of cell death but also in limiting the damage-associated inflammation.     

Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC(50) of 1 microm. It did not inhibit acid SMase at up to at least 150 microm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 microm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 microm of the compound was protected completely from the loss of SM. The addition of 10-20 microm GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 microm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 microm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-kappaB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.     

Distinct signaling pathways in TRAIL- versus tumor necrosis factor-induced apoptosis

Trimeric tumor necrosis factor (TNF) binding leads to recruitment of TRADD to TNFR1. In current models, TRADD recruits RIP, TRAF2, and FADD to activate NF-kappaB, Jun N-terminal protein kinase (JNK), and apoptosis. Using stable short-hairpin RNA (shRNA) knockdown (KD) cells targeting these adaptors, TNF death-inducing signaling complex immunoprecipitation demonstrates competitive binding of TRADD and RIP to TNFR1, whereas TRAF2 recruitment requires TRADD. Analysis of KD cells indicates that FADD is necessary for Fas-L- or TRAIL- but not TNF-induced apoptosis. Interestingly, TRADD is dispensable, while RIP is required for TNF-induced apoptosis in human tumor cells. TRADD is required for c-Jun phosphorylation upon TNF exposure. RIP KD abrogates formation of complex II following TNF exposure, whereas TRADD KD allows efficient RIP-caspase 8 association. Treatment with TRAIL also induces formation of a complex II containing FADD, RIP, IKKalpha, and caspase 8 and 10, leading to activation of caspase 8. Our data suggest that TNF triggers apoptosis in a manner distinct from that of Fas-L or TRAIL.     

Butylated hydroxyanisole specifically inhibits tumor necrosis factor-induced cytotoxicity and growth enhancement.

The effect of commonly used food antioxidants on recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-alpha-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-alpha-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NF kappa B after 10 or 60 min challenge with rTNF-alpha in L929 cells. BHA also inhibited rTNF-alpha-induced, but not rIL-1 beta-induced growth enhancement in FS-4 fibroblasts. Further, BHA blocked both rTNF-alpha-induced and rIL-1 beta-induced prostaglandin E2 synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-alpha-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither alpha-tocopherol nor BHA inhibited rTNF-alpha-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-alpha- and rTNF-beta-induced cytotoxicity, as well as of rTNF-alpha-induced growth enhancement.     

Effects of thromboxane synthase inhibition on tumor necrosis factor-induced lung injury in sheep.

To examine the role of thromboxane (Tx) A2 in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we tested the effects of OKY-046, a selective thromboxane synthase inhibitor, on pulmonary hemodynamics, lung lymph balance, circulating leukocytes, arterial blood gas analysis, and TxA2 (as TxB2) and prostacyclin (as 6-keto-prostaglandin F1 alpha) levels in plasma and lung lymph after TNF infusion in awake sheep. Infusion of human recombinant TNF (3.5 micrograms/kg) into a chronically instrumented awake sheep caused a transient increase in pulmonary arterial pressure (Ppa). The Ppa peaked within 15 min of the start of TNF infusion from 23.3 +/- 1.1 (SE) cmH2O of baseline to 42.3 +/- 2.3 cmH2O and then decreased toward baseline. The pulmonary hypertension was accompanied by transient hypoxemia, peripheral leukopenia, and the increases in TxB2 in plasma and lung lymph. These changes were followed by an increase in flow of protein-rich lung lymph, consistent with an increase in pulmonary microvascular permeability. OKY-046 significantly prevented the rises of Ppa and TxB2 concentrations in plasma and lung lymph during early phase after TNF infusion. OKY-046, however, did not attenuate the increase of lung lymph flow, transient hypoxemia, and leukopenia. From these data, and by comparison with our previous studies of OKY-046-pretreated sheep during endotoxemia, we conclude that TxA2 has an important role of the increase in the early pulmonary hypertension, but it is not related to the early hypoxemia, leukopenia, and lung lymph balances in TNF-induced lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb

Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.     

Isolation and characterization of eight tumor necrosis factor-induced gene sequences from human fibroblasts.

A lambda cDNA library was prepared from polyadenylated RNA isolated from quiescent human diploid FS-4 fibroblasts stimulated with tumor necrosis factor for 3 h. Differential screening was used to isolate cDNA sequences that are stimulated by tumor necrosis factor. Eight distinct tumor necrosis factor-stimulated gene sequences (designated TSG-1, -6, -8, -12, -14, -21, -27, and -37) were partially sequenced and compared with known sequences from GenBank. TSG-1 was identical to the gene for interleukin-8. TSG-8 corresponded to the gene for monocyte chemotactic and activating factor. TSG-21 and -27 were identical to the genes for collagenase and stromelysin, respectively. The other four sequences showed no homologies with known genes. Patterns of induction of mRNAs corresponding to the eight cloned cDNAs by various cytokines, growth factors, and activators of second messenger pathways were analyzed in FS-4 cells.     

Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma

Summary Epigenetic models for tumor formation assume that oncogenic transformation results from changes in the activity of otherwise normal genes. Since gene activity can be inhibited by DNA methylation, and inactivation of tumor suppressor genes is a fundamental process in oncogenesis, we investigated the methylation status of the retinoblastoma suppressor gene (RB gene) on chromosome 13, in blood and tumor cells from 21 retinoblastoma patients. Using methylation-sensitive restriction enzymes and a cloned DNA probe for the unmethylated CpG island at the 5 end of RB gene, we obtained evidence of hypermethylation of this gene in a sporadic unilateral retinoblastoma tumor. The closely linked esterase D gene and a CpG-rich island on chromosome 15 were not affected. We suggest that changes in the methylation pattern of the RB gene play a role in the development and spontaneous regression of some retinoblastoma tumors.     

Requirement of FADD for tumor necrosis factor-induced activation of acid sphingomyelinase

The generation of mice strains deficient for select members of the signaling complex of the 55-kDa tumor necrosis factor receptor (TNF-R55) has allowed the assignment of specific cellular responses to distinct TNF-R55-associated proteins. In particular, the TNF-R55-associated protein FADD seems to be responsible for recruitment and subsequent activation of caspase 8. In this report we demonstrate the requirement of FADD for TNF-induced activation of endosomal acid sphingomyelinase (A-SMase). In primary embryonic fibroblasts from FADD-deficient mice the activation of A-SMase by TNF-R55 ligation was almost completely impaired. This effect is specific in that other TNF responses like activation of NF-kappaB or neutral (N-)SMase remained unaffected. In addition, interleukin-1-induced activation of A-SMase in FADD-deficient cells was unaltered. In FADD-/- embryonic fibroblasts reconstituted by transfection with a FADD cDNA expression construct, the TNF responsiveness of A-SMase was restored. The results of this study suggest that FADD, in addition to its role in triggering a proapoptotic caspase cascade, is required for TNF-induced activation of A-SMase.     

Inhibition of tumor necrosis factor-induced cell killing by tryptophan and indole

Cells sensitive to the cytocidal effect of tumor necrosis factor (TNF) were protected against this effect when growth in the presence of elevated concentrations of tryptophan. Several other indole derivatives also provided protection against TNF cytotoxicity. Most effective were indole itself and its monomethyl derivatives, providing a degree of protection greatly exceeding that observed with tryptophan. Protection was also observed against the cytocidal effect of TNF applied in the presence of a protein synthesis inhibitor. The protective effect of tryptophan was largely dependent on preexposure of the cells, for several hours, to a high concentration of this amino acid. On the other hand, indole was protective also when applied to cells together with TNF, or even two hours after TNF application. The inhibition of the cytotoxicity of TNF by tryptophan and other indole derivatives may serve as a useful experimental tool in exploring the mechanisms and the physiological implications of TNF cytotoxicity.