The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.

Resistance against the cytotoxic actions of tumor necrosis factor alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.     

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.     

Effect of pyridoxine on tumor necrosis factor activitiesin vitro

Clinical trials with tumor necrosis factor (TNF) as an antitumor agent have so far given rather disappointing results. In this study we show that the naturally occuring vitamin B₆ compound, pyridoxine, enhances TNF-induced cytolysis of three subclones of a mouse fibrosarcoma cell line (WEHI 164). The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to be dependent on the cells sensitivity to TNF, as the enhancement was much more pronounced in the relatively TNF resistant subclone act-R(cl.12)-WEHI 164, than in the very TNF sensitive subclone WEHI 164 clone 13. Furthermore, our study shows that pyridoxine, in contrast to its enhancing effect on TNF-induced cytotoxicity, rather inhibits TNF-induced growth of human FS-4 fibroblasts. Pyridoxine also enhances lymphotoxin (LT)-induced tumor cell killing and inhibits LT-induced fibroblast growth. Pyridoxine is a relatively non-toxic agentin vivo. Our results suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone, in the treatment of cancer patients.     

The p70 tumor necrosis factor receptor mediates cytotoxicity.

Tumor necrosis factor alpha (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface receptors (TNFRs), proteins of 55 kd (p55) and 70-80 kd (p70), mediate TNF action. Mouse TA1 cells are not killed by human (h) TNF, but are killed by mouse (m) TNF alone. Since the mouse p70 TNFR is recognized only by mTNF, these results implicate p70 receptor action in TA1 cell killing. Human HeLa cells have mainly the p55 receptor and are not killed by hTNF alone. When transfected with the human p70 TNFR, HeLa p70 die within 24 hr. HeLa p70 cells also show reduced c-fos and manganous superoxide dismutase induction by TNF. NIH 3T3 mouse fibroblasts are sensitive to only mTNF, but overexpression of the human p70 receptor causes cell death by hTNF and increased sensitivity to mTNF. These results provide a direct function for the p70 TNFR in TNF-induced cytotoxicity.     

Activation of the insulin-like growth factor-I receptor inhibits tumor necrosis factor-induced cell death

The effect of insulin-like growth factor (IGF) on tumor necrosis factor (TNF)-induced cell killing was determined for mouse BALB/c3T3 fibroblasts in vitro. Cells maintained in 0.5% fetal bovine serum (FBS) were killed by TNF within 6 h in a concentration-dependent manner, an effect that was prevented by IGF-I. TNF-induced cytotoxicity of 3T3 cells that overexpress the human IGF-I receptor (p6 cells) was prevented by IGF-I alone in the absence of serum. TNF-induced cell death was associated with the morphologic features of apoptosis and the release of low-molecular-weight DNA, both of which were prevented by IGF-I. Neither epidermal growth factor (EGF) nor platelet-derived growth factor (PDGF) protected p6 cells from TNF-induced apoptosis. The specific protective action of the IGF-I receptor was demonstrated further by the marked sensitivity to TNF of embryo fibroblasts derived from mice with targeted disruption of the IGF-I receptor (R cells) but not of fibroblasts derived from wild-type littermates or R cells transfected with the cDNA for the human IGF-I receptor. Cycloheximide or actinomycin D markedly reduced the protection offered by IGF-I. IGF-I protection of BALB/c3T3 cells persisted for up to 5 days in the presence of PDGF and EGF, whereas IGF-I lost its effectiveness after 2 days in the absence of growth factors. IGF-I did not prevent TNF-induced release of arachidonic acid. The results demonstrate a specific role for the IGF-I receptor in the protection against TNF cytotoxicity. This action of the IGF-I receptor is mediated by protective cytosolic proteins that exhibit a high rate of turnover and whose levels are regulated principally by factors within serum other than IGF-I. © 1996 Wiley-Liss, Inc.     

c-Myc sensitizes cells to tumor necrosis factor-mediated apoptosis by inhibiting nuclear factor kappa B transactivation

Nuclear factor kappaB (NF-kappaB) plays a key role in suppression of tumor necrosis factor (TNF)-mediated apoptosis by inducing a variety of anti-apoptotic genes. Expression of c-Myc has been shown to sensitize cells to TNF-mediated apoptosis by inhibiting NF-kappaB activation. However, the precise step in the NF-kappaB signaling pathway and apoptosis modified by c-Myc has not been identified. Using the inducible c-MycER system and c-Myc null fibroblasts, we found that expression of c-Myc inhibited NF-kappaB activation by interfering with RelA/p65 transactivation but not nuclear translocation of NF-kappaB. Activation of c-Myc promoted TNF-induced release of cytochrome c from mitochondria to the cytosol because of the inhibition of NF-kappaB. Furthermore, we found that NF-kappaB-inducible gene A1 was attenuated by expression of c-Myc and that the restoration of A1 expression suppressed c-Myc-induced TNF sensitization. Our results elucidate the molecular mechanisms by which c-Myc increases cell susceptibility to TNF-mediated apoptosis, indicating that c-Myc may exhibit its pro-apoptotic activities by repression of cell survival genes.     

Redox regulation of TNF signaling

TNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF-treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF-induced ROS as compared to pro-oxidant-induced ROS we conclude that TNF-mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron-transport inhibitors, glutathione and thiol-reactive agents but also caspase inhibitors modulate TNF-induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF-induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.     

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.     

Tumor necrosis factor is cytotoxic to human fibroblasts in the presence of exogenous arachidonic acid

Recombinant human tumor necrosis factor (TNF) stimulated the growth of confluent human fibroblasts (FS-4) in serum-free culture medium. However, TNF had a cytotoxic effect upon the growth of FS-4 cells in combination with arachidonic acid. When arachidonic acid was added to culture medium in the absence of TNF, however, it had no effect on the cell growth. Arachidonic acid inhibited the TNF-induced cell growth in a dose-dependent manner: it reversed the TNF-stimulated growth to the control level at a concentration of 10 microM and was cytotoxic to TNF-treated FS-4 cells at higher concentrations. This cytotoxicity of TNF was not observed in FS-4 cells treated with palmitic acid. Indomethacin, a cyclooxygenase inhibitor, decreased the cytotoxic effect that TNF exerted in the presence of arachidonic acid. These results suggest that TNF becomes cytotoxic to FS-4 cells when arachidonic acid present in the culture medium is converted to prostaglandins.     

Lithium sensitizes tumor cells in an NF-kappa B-independent way to caspase activation and apoptosis induced by tumor necrosis factor (TNF). Evidence for a role of the TNF receptor-associated death domain protein

We have previously shown that lithium salts can considerably increase the direct cytotoxic effect of tumor necrosis factor (TNF) on various tumor cells in vitro and in vivo. However, the underlying mechanism has remained largely unknown. Here we show that the TNF-sensitizing effect of lithium chloride (LiCl) is independent of the type of cell death, either necrosis or apoptosis. In the case of apoptosis, TNF/lithium synergism is associated with an enhanced activation of caspases and mitochondrial cytochrome c release. Sensitization to apoptosis is specific for TNF-induced apoptosis, whereas Fas-mediated or etoposide-induced apoptosis remains unaffected. LiCl also potentiates cell death induced by artificial oligomerization of a fusion protein between FKBP and the TNF receptor-associated death domain protein. TNF-induced activation of NF-kappa B-dependent gene expression is not modulated by LiCl treatment. These results indicate that LiCl enhances TNF-induced cell death in an NF-kappa B-independent way, and suggest that the TNF receptor-associated death domain protein plays a crucial role in the TNF-sensitizing effect of LiCl.     

Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.     

Gelsolin, but not its cleavage, is required for TNF-induced ROS generation and apoptosis in MCF-7 cells

The tumor necrosis factor (TNF) can induce apoptosis in many cells including MCF-7 cells. To identify the genes responsible for TNF-induced apoptosis, we generated a series of TNF-resistant MCF-7 cell lines by employing retrovirus insertion-mediated random mutagenesis. In one of the resistant lines, gelsolin was found to be disrupted by viral insertion. Exogenous expression of gelsolin in this mutant cell line (Gel^mut) restored the sensitivity to TNF-induced cell death and knock-down of gelsolin by siRNA conferred MCF-7 cells with resistance to TNF, indicating that gelsolin is required for TNF-induced apoptosis. Interestingly, the resistance of Gel^mut cells to apoptosis induction is selective to TNF, since Gel^mut and wild-type cells showed similar sensitivity to other death stimuli that were tested. Furthermore, TNF-induced ROS production in Gel^mut cells was significantly decreased, demonstrating that gelsolin-mediated ROS generation plays a crucial role in TNF-induced apoptosis in MCF-7 cells. Importantly, caspase-mediated gelsolin cleavage is dispensable for TNF-triggered ROS production and subsequent apoptosis of MCF-7 cells. Our study thus provides genetic evidence linking gelsolin-mediated ROS production to TNF-induced cell death.     

Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.     

Mannose 6-phosphorylated proteins are required for tumor necrosis factor-induced apoptosis: defective response in I-cell disease fibroblasts

Whereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF.     

Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se.

L929, a murine fibrosarcoma cell line highly sensitive to the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF), was used as a target cell in our studies. We [Suffys et al. (1987) Biochem. Biophys. Res. Commun. 149, 735-743], as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A2-type enzyme, is likely to be involved in TNF-mediated cell killing. We now further document this conclusion and provide suggestive evidence that the enzyme activity specifically involved in TNF cytotoxicity differs from activities associated with the eventual cell death process itself or with non-toxic serum treatment. We also show that the 5,8,11,14-icosatetraenoic acid (arachidonic acid, delta 4 Ach) released by PL, and possibly metabolized, is unlikely to be a key mediator of the TNF-mediated cytotoxicity. These conclusions are based on the following experimental findings. 1. TNF treatment of cells, prelabelled for 24 h with [3H] delta 4Ach or [14C] delta 3Ach (delta 3Ach identical to 5,8,11-icosatrienoic acid) resulted in an early, time-dependent and concentration-dependent release of radioactivity in the supernatant preceding actual cell death. The extent of this response was moderate, albeit reproducible and significant. Analysis of the total lipid fraction from cells plus supernatant revealed that only release of arachidonic acid from phospholipids, but not its metabolization was induced by TNF. However, the release of less unsaturated fatty acids, such as linoleic acid (Lin) or palmitic acid (Pam), was not affected during the first hours after TNF addition. 2. An L929 subclone, selected for resistance to TNF toxicity, was found to be defective in TNF-induced delta 4Ach libration. 3. Interleukin-1 (IL1) was not cytotoxic for L929 and did not induce release of delta 4Ach. 4. Release of delta 4Ach was not restricted to TNF; the addition of serum to the cells also induced release of fatty acids into the medium. In this case, however, there was no specificity, as all fatty acids tested, including Lin and Pam, were released. 5. Inhibition of PL-A2 activity by appropriate drugs markedly diminished TNF-induced delta 4Ach release and resulted also in a strong decrease in TNF-induced cytotoxicity. 6. Other drugs, including serine protease inhibitors, which strongly inhibit TNF-induced cytotoxicity, also decreased the TNF-induced delta 4Ach release, whereas LiCl potentiated both TNF-mediated effects. 7. Protection of cells against TNF toxicity by means of various inhibitors was not counteracted by addition of exogenous fatty acids, including delta 4Ach.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.