TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated IkappaBalpha degradation and NF-kappaB nuclear translocation in L929 fibroblasts

The role of transforming growth factor beta1 (TGF-beta1)-induced extracellular matrix proteins in the modulation of cellular response to the cytotoxic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Murine L929 fibroblasts were prestimulated with or without TGF-beta1 for 1-24 h and the resulting extracellular protein matrices were prepared. Unstimulated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta1 matrix-stimulated cells resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF resistance is independent of the NF-kappaB antiapoptotic effect. For example, exposure of TGF-beta1 matrix-stimulated L929 cells to TNF failed to result in IkappaBalpha degradation and NF-kappaB nuclear translocation or activation. Also, control matrix stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta1 matrix-stimulated cells. By metabolic labeling, ammonium sulfate precipitation and N-terminal amino acid microsequencing, TGF-beta1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptide antibodies against its N-terminus removed the TGF-beta1 matrix protein-mediated protection against TNF/ActD cytotoxicity and its enhancement of anti-Fas/ActD killing, indicating that p46 is responsible for these effects. Immunostaining of L929 cells revealed that the antibodies were bound to a membrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived from the released membrane p100.     

Tumor necrosis factor-mediated cytotoxicity involves ADP-ribosylation

The mechanism of TNF-mediated cytotoxicity was studied in several cell lines, including L929 murine fibroblasts. TNF caused a time- and dose-dependent increase of ADP-ribosylation in L929 target cells parallel to cell death. During the course of TNF-mediated cytotoxicity in the presence of actinomycin D, an increase in ADP-ribosylation became apparent between 4 and 6 h after exposure to TNF. Intracellular NAD+ and ATP levels decreased parallel to but not preceding cell death. Two inhibitors of ADP-ribosylation, namely 3-aminobenzamide and nicotinamide, prevented TNF-mediated cytotoxicity. Another target, the human cervical carcinoma cell line ME-180, showed an increase in ADP-ribosylation when treated with TNF, and the cytotoxic action of TNF on this target cell was inhibited by these two inhibitors. In the absence of actinomycin D, treatment of L929 cells with TNF also increased ADP-ribosylation, and the cytotoxic action of TNF was inhibited by nicotinamide. These results indicate that ADP-ribosylation may be involved in the TNF-mediated cytotoxic reaction.     

Glutathione S-transferases as antioxidant enzymes: small cell lung cancer (H69) cells transfected with hGSTA1 resist doxorubicin-induced apoptosis

It has been suggested that the alpha-class glutathione S-transferases (GSTs) protect various cell types from oxidative stress and lipid peroxidation (LPO). In order to examine the protective role of alpha-class GST isozyme hGSTA1-1 against doxorubicin (DOX)-induced lipid peroxidation, cytotoxicity, and apoptosis, human small cell lung cancer (SCLC) H69 cells were stably transfected with hGSTA1. Immunological and biochemical characterization of hGSTA1-transfected cells revealed the expression of functionally active hGSTA1-1 localized near the cellular plasma membranes. hGSTA1-transfected cells acquired significantly increased resistance to the DOX-induced cytotoxicity by suppressing lipid peroxidation levels in these cells. Overexpression of hGSTA1-1 in cells inhibited DOX-mediated depletion of GSH and higher GSH levels were found in DOX-treated hGSTA1-transfected cells as compared with empty vector-transfected controls. hGSTA1-1 overexpression also provided protection to cells from DOX-induced apoptosis by inhibiting phosphorylation of c-Jun-N-terminal kinases (JNK), caspase-3 activation, and by preserving the levels of anti-apoptotic protein Bcl-2. These results are consistent with the idea that the alpha-class GSTs provide protection against oxidative stress by attenuating lipid peroxidation and these enzymes can modulate signaling for apoptosis.     

Reduced expression of manganese superoxide dismutase in cells resistant to cytolysis by tumor necrosis factor.

Tumor necrosis factor (TNF) induces synthesis of manganese superoxide dismutase (MnSOD). It was previously shown that overexpression of MnSOD protected some mammalian cells from TNF cytotoxicity. The purpose of this study was to establish whether MnSOD was increased in cells selected for resistance to cytolysis by TNF in combination with cycloheximide. Melanoma SK-MEL-109 and HeLa cell-resistant variants were selected by repeated treatments with TNF and cycloheximide. The SK-MEL-109 variants had relatively low levels of MnSOD that were inducible by TNF. Surprisingly, the HeLa variants had very low levels of MnSOD that were poorly inducible by either TNF or interleukin-1 alpha. Therefore, an elevated level of MnSOD was not required to protect these cells from TNF-mediated cytolysis. The HeLa variants were more sensitive than parental cells to superoxide radical (O2-) generating compounds, such as paraquat or xanthine/xanthine oxidase. Pretreatment of these variants with TNF did not provide protection against damage by superoxide radicals.     

HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.     

Hyaluronidase activation of c-Jun N-terminal kinase is necessary for protection of L929 fibrosarcoma cells from staurosporine-mediated cell death

Hyaluronidase counteracts the growth inhibitory function of transforming growth factor beta (TGF-beta), whereas secretion of autocrine TGF-beta and hyaluronidase is necessary for progression and metastasis of various cancers. Whether hyaluronidase and TGF-beta1 induce resistance to staurosporine in L929 fibrosarcoma cells was investigated. When pretreated with TGF-beta1 for 1-2 h, L929 cells resisted staurosporine apoptosis. In contrast, without pretreatment, hyaluronidase protected L929 cells fromstaurosporine apoptosis. Hyaluronidase rapidly activated p42/44 MAPK (or ERK) in L929 cells and TGF-beta1 retarded the activation. Nonetheless, TGF-beta1 synergistically increased hyaluronidase-mediated inhibition of staurosporine apoptosis. Hyaluronidase rapidly activated c-Jun N-terminal kinase (JNK1 and JNK2) in L929 cells in 20 min. Dominant negative JNK1, JNK2, and JNK3 abolished the hyaluronidase inhibition of staurosporine apoptosis, but not the TGF-beta1 protective effect. Unlike the resistance to staurosporine, pretreatment of L929 cells with hyaluronidase is necessary to generate resistance to other anticancer drugs, including doxorubicin, daunorubicin, actinomycin D, and camptothecin, and the induced resistance was also blocked by dominant-negative JNKs. Together, hyaluronidase-mediated JNK activation is necessary to generate resistance to various anticancer drugs in L929 cells.     

IkappaBalpha is essential for maintaining basal c-Jun N-terminal kinase (JNK) activation and regulating JNK-mediated resistance to tumor necrosis factor cytotoxicity in L929 cells.

Early activation of c-Jun N-terminal kinase (JNK) is believed to block apoptosis in response to death signals such as tumor necrosis factor (TNF). Brief exposure of murine L929 fibroblasts to anisomycin for 1 hr to activate JNK resulted in resistance to TNF killing. TNF rapidly induced cytoplasmic shrinkage in control cells, but not in the anisomycin-pretreated L929 cells. However, the induced TNF resistance was suppressed in the L929 cells which were engineered to stably inhibit IkappaBalpha protein expression by antisense mRNA ( approximately 80% reduction in protein expression). No constitutive NF-kappaB nuclear translocation and increased TNF resistance were found in these IkappaBalpha antisense cells. Notably, these cells had a significantly reduced basal level of JNK activation (50-70%), compared to vector control cells. Furthermore, brief exposure of L929 cells to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), resulted in resistance to TNF killing, probably due to preconsumption of caspases by wortmannin. Nonetheless, wortmannin-induced TNF resistance was suppressed in the IkappaBalpha antisense cells. Thus, these observations indicate that IkappaBalpha is essential for maintaining the basal level of JNK activation and regulating the JNK-induced TNF resistance.     

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

Synergistic interactions between tumor necrosis factor and inhibitors of DNA topoisomerase I and II

TNF is a pleiotropic cytokine that mediates diverse cellular responses, including cytotoxicity, cytostasis, proliferation, differentiation, and the expression of specific genes. Many of these processes require the activity of DNA topoisomerases I and II. We have investigated the interactions of TNF with inhibitors of both topoisomerases in 16-h assays using the murine L929 and human ME-180 cell lines, which undergo a cytotoxic TNF response. Camptothecin, a specific inhibitor of topoisomerase I, enhanced TNF cytotoxicity 150-fold against both cell lines. The topoisomerase II inhibitors VM-26 and VP-16, which stabilize covalent DNA-topoisomerase intermediates, greatly enhance TNF cytotoxicity against both cell lines. The most effective, VM-26, can lower the TNF LD50 to femtomolar levels. In contrast, the topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in ME-180 cells. The large changes in TNF sensitivity induced by drug concentrations that by themselves show no effect, and the opposing synergistic effects of inhibitors with different inhibitory mechanisms (in L929 cells), suggest the active involvement of topoisomerases in TNF-mediated cytotoxicity. The correlation of cytotoxic synergy with the stabilization of DNA strand breaks indicates that DNA damage may play a significant role in TNF-mediated cytotoxicity.     

Suppression of IkappaBalpha expression is necessary for c-Jun N-terminal kinase-mediated enhancement of Fas cytotoxicity

The role of c-Jun N-terminal kinase (JNK) in the regulation of Fas-mediated cell death was investigated. Murine L929 fibroblasts were pretreated with anisomycin for 1 h to activate JNK, followed by exposure to anti-Fas antibodies/actinomycin D (ActD) for 16-24 h. Compared to untreated controls, the induction of JNK activation failed to raise cellular sensitivity to anti-Fas/ActD killing. Notably, a significant increase in anti-Fas/ActD killing as induced by JNK preactivation was observed in L929 cells which were engineered to suppress IkappaBalpha protein expression by antisense mRNA. Restoration of the IkappaBalpha protein level in these cells by ectopic expression of a cDNA construct abolished the JNK-increased anti-Fas/ActD killing. Despite the suppression of IkappaBalpha, no constitutive p65 (RelA) NF-kappaB nuclear translocation was observed in the IkappaBalpha-antisense cells. Also, inhibition of NF-kappaB by curcumin failed to inhibit the JNK-increased Fas cytotoxicity, suggesting that NF-kappaB is not involved in the observed effect. Most interestingly, culturing of L929 cells on extracellular protein matrices resulted in partial suppression of IkappaBalpha expression and constitutive JNK and p42/44 MAPK activation. Upon stimulation with anisomycin, these matrix protein-stimulated cells further exhibited reduced IkappaBalpha expression and p42/44 MAPK activation, as well as became sensitized to JNK-increased anti-Fas/ActD killing. Again, ectopic expression of IkappaBalpha in these cells abolished the enhanced anti-Fas/ActD killing effect. Together, these results indicate that suppression of IkappaBalpha expression is essential for JNK-mediated enhancement of Fas cytotoxicity.     

Purification and biologic characterization of a specific tumor necrosis factor alpha inhibitor

The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.     

Possible cytotoxic mechanisms of TNF in vitro

TNF is a protein originally isolated by its ability to induce hemorrhagic necrosis of some tumors in vivo, and cytotoxic effects against certain tumor cells, but not against normal cells in vitro. Though mechanisms of the cytotoxicity and selectivity of TNF action in vitro are not clear, there is much evidence that free radicals such as superoxide and hydroxyl radicals, mediated by TNF, are correlated with TNF cytotoxicity. Recently, manganous superoxide dismutase (MnSOD) in mitochondria has been demonstrated to be a rescue protein against TNF cytotoxicity and to be induced when the cells are exposed to TNF in the resistant cells. Two steroid hormones, glucocorticoid and 1,25-dihydroxyvitamin D3, block the cytotoxicity of TNF. Glucocorticoid may reduce TNF cytotoxicity by inhibiting phospholipase A2 and 1,25-dihydroxyvitamin D3 by inducing MnSOD in combination with TNF.     

Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region

Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.     

Antioxidant defenses in the TNF-treated MCF-7 cells: selective increase in MnSOD

Oxidative stress has been implicated in the mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis, raising a question about the status of antioxidant defenses in TNF-sensitive cells. Antioxidant defenses were examined in MCF-7 cells after treatment with TNF. Cell morphology and DNA fragmentation assays were used to confirm increased apoptosis as a result of TNF treatment. The expression and activity of antioxidant defenses were assessed using Northern blot hybridization analyses and biochemical assays, respectively. Five- and ten-fold increases in manganese superoxide dismutase (MnSOD) mRNA were measured after one and five days of TNF treatment, respectively. The expression of copper,zinc superoxide dismutase, catalase or thioredoxin was not altered. An approximate five-fold increase in MnSOD activity followed the change in gene expression, but no difference in the activity of catalase or glutathione peroxidase was seen. Thus, increased MnSOD activity was not accompanied by an increase in other antioxidant defenses and in particular, H2O2-scavenging enzymes. MnSOD has previously been shown to afford protection against TNF-mediated cytotoxicity. The observed lack of increased peroxidase activity is consistent with mitochondrially-generated superoxide anion radical contributing to the mechanism of TNF-induced apoptosis.     

Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells

Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.     

Bcl-2 inhibits tumor necrosis factor-alpha-mediated increase of glycolytic enzyme activities and enhances pyruvate carboxylase activity

To understand the effects of bcl-2 on glucose metabolism and tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity, the activities of glycolytic enzymes (hexokinase, 6-phosphofructo-1-kinase, and pyruvate kinase), lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were examined with or without TNF-alpha treatment in TNF-alpha sensitive L929 cells and TNF-alpha resistant bcl-2 transfected L929 cells. In TNF-alpha-treated L929 cells, the activities of the glycolytic enzymes and lactate dehydrogenase greatly increased, but there was no detectable change in phosphoenolpyruvate carboxykinase. Pyruvate carboxylase activity decreased by about 25% between 6 and 12 h after TNF-alpha treatment. The activities of the glycolytic enzymes and lactate dehydrogenase in bcl-2 transfected L929 cells were lower than in L929 cells upon TNF-alpha treatment. On the other hand, the activity of pyruvate carboxylase was 20-100% greater after 6 h of TNF-alpha treatment than in the L929 cells. The activity of phosphoenolpyruvate carboxykinase of bcl-2 trasfected L929 cells was lower by up to 25% than in L929 cells after 12 h. The increase of pyruvate carboxylase activity and decrease of phosphoenolpyruvate carboxykinase activity in bcl-2 transfected L929 cells may contribute to the protective effects of bcl-2 against TNF-alpha mediated cytotoxicity.     

Hypoxia and resistance to hydrogen peroxide confer resistance to tumor necrosis factor in murine L929 cells.

The mechanism whereby tumor necrosis factor (TNF) kills mammalian cells is not well understood, although oxidative damage has been suggested by several investigators. Further, it is not known why cells vary in their responsiveness to TNF. We show that the cytotoxic effect of TNF toward TNF-sensitive L929 cells is blocked under hypoxic conditions, suggesting a critical role of molecular oxygen and reactive oxygen species. To test whether cellular resistance to reactive oxygen species could provide resistance to TNF, we derived a variant strain from L929 cells by chronic exposure to an oxidizing agent, hydrogen peroxide (H2O2). These cells exhibit marked resistance to TNF as well as to H2O2. This cross-protection provides additional evidence that mechanisms of resistance to oxidative damage are causally related to TNF-induced cell death. Scatchard analysis of TNF binding did not reveal significant differences between the H2O2-resistant line and the wild-type L929 line. On the other hand, analyses of antioxidant enzymes and glutathione levels in cells of the wild-type and the H2O2-resistant lines revealed several potentially important differences. Before exposure to TNF, the H2O2-resistant variants have elevated catalase activity, decreased activity of total glutathione-S-transferase (GST), and similar superoxide dismutase (SOD) activities. Exposure to TNF led to alteration in CuZnSOD activity, and much more so in the variants than in the wild-type L929 cells. However, no significant change in MnSOD activities in cells of either cell line was observed. Total GST activity was not altered appreciably by TNF in either cell line, but Western analysis showed that the level of alpha GST isozyme was increased and mu GST isozyme decreased in the H2O2-resistant variants. Furthermore, alterations in total glutathione content were observed in both the control and the variant cells.     

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.