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.     

Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.     

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.     

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.     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.     

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.     

Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.     

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation

The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [(3)H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA(2) (sPLA(2)) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA(2) or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA(2) (cPLA(2)) assayed in vitro as well as increased monocyte tumor necrosis factor-alpha secretion. The AA liberation was attenuated by inhibitors toward cPLA(2) and sPLA(2), indicating that both PLA(2) enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA(2) or SMase potentiates cellular AA release and cPLA(2) activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA(2) and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.     

Resistance to Tumor Necrosis Factor-Induced Cell Death Mediated by PMCA4 Deficiency

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor (TNF)-resistant lines from L929 cells. Using this approach, we discovered that the plasma membrane calcium ATPase 4 (PMCA4) is required for TNF-induced cell death in L929 cells. Under basal conditions, PMCA4-deficient (PMCA(mut)) cells have a normal phenotype. However, stimulation with TNF induces an abnormal increase in the intracellular calcium concentration ([Ca(2+)](i)). The substantially elevated [Ca(2+)](i) caused resistance to TNF-induced cell death. We found that an increase in the total volume of acidic compartments (VAC), mainly constituted by lysosomes, is a common event in cell death caused by a variety of agonists. The increased [Ca(2+)](i) in PMCA(mut) cells promoted lysosome exocytosis, which, at least in part, accounted for the inhibition of TNF-induced increase in VAC and cell death. Promoting lysosome exocytosis by calcium inhibited TNF-induced cell death in wild-type L929 cells, while inhibition of lysosome exocytosis or increase of VAC by sucrose restored the sensitivity of PMCA(mut) cells to TNF-induced cell death. Thus, increase of the volume of acidic compartment is a part of the cell death process, and the antideath effect of calcium is mediated, at least in part, by inhibition of the TNF-induced increase in VAC.     

Arachidonic acid metabolism via cytosolic phospholipase A2 α induces cytotoxicity in niemann-pick disease type C cells

Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E(2) compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A(2) α (cPLA(2) α) and cultivation in lipoprotein-deficient medium. There was no difference in the expression of both cyclooxygenase-1 and -2 between NPC cells and wild-type cells. U18666A, a cholesterol transport-inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A-induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA(2) α knockdown L929 cells. These results suggest that cPLA(2) α-AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells.     

Tumor necrosis factor-induced microtubule stabilization mediated by hyperphosphorylated oncoprotein 18 promotes cell death

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser(16) and Ser(63) are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.     

Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells

Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.     

Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.     

Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells

Recombinant tumor necrosis factor alpha (rTNF-alpha)-induced release of endogenous fatty acids was examined in WEHI 164 clone 13 fibrosarcoma cells using a highly sensitive HPLC method. The initial rTNF-alpha-induced extracellular release of endogenous fatty acids was dominated by 20:4n;-6, 22:4n;-6, 24:4n;-6, and 18:1n;-9 showing relative rates of 2.9, 0.9, 1.1, and 1.0, respectively. Release of endogenous AA and DNA fragmentation occurred simultaneously and preceded cell death by approx. 2 h. Methyl arachidonoyl fluorophosphonate and LY311727, specific inhibitors of Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)), respectively, neither blocked rTNF-alpha-induced cytotoxicity or endogenous AA release. However, both inhibitors reduced rTNF-alpha-induced release of other endogenous fatty acids. In comparison, the antioxidant butylated hydroxyanisole (BHA) completely inhibited the rTNF-alpha-induced cytotoxicity as well as AA release mediated through the TNF receptor p55, while the very similar antioxidant butylated hydroxytoluene had no effect. BHA did not inhibit recombinant cPLA(2) or sPLA(2) enzyme activity in vitro. Furthermore, stimulation of cells with rTNF-alpha for 4 h did not increase cPLA(2) enzyme activity. The data indicate that neither cPLA(2) or sPLA(2) mediate rTNF-alpha-induced apoptosis and extracellular AA release in WEHI cells. The results suggest that a BHA-sensitive signaling pathway coupled to AA release is a key event in TNF-induced cytotoxicity in these cells.     

Effect of tumor necrosis factor on GTP binding and GTPase activity in HL-60 and L929 cells

Tumor necrosis factor (TNF) is a monokine that induces pleiotropic events in both transformed and normal cells. These effects are initiated by the binding of TNF to high affinity cell surface receptors. The post-receptor events and signaling mechanisms induced by TNF, however, have remained unknown. The present studies demonstrate the presence of a single class of high affinity receptors on membranes prepared from HL-60 promyelocytic leukemic cells. The interaction of TNF with these membrane receptors was associated with a 3.8-fold increase in specific binding of the GTP analogue, GTP gamma S. Scatchard analysis of GTP gamma S binding data demonstrated that TNF stimulates GTP binding by increasing the affinity of available sites. The TNF-induced stimulation of GTP binding was also associated with an increase in GTPase activity. Moreover, the increase in GTPase activity induced by TNF was sensitive to pertussis toxin. The results also demonstrate that TNF similarly increased GTP binding and pertussis toxin-sensitive GTPase activity in membranes from mouse L929 fibroblasts, thus indicating that these effects are not limited to hematopoietic cells. Analysis of HL-60 membranes after treatment with pertussis toxin in the presence of [32P]NAD revealed three substrates with relative molecular masses of approximately Mr 41,000, 40,000, and 30,000. In contrast, L929 cell membranes had only two detectable pertussis toxin substrates of approximately Mr 41,000 and 40,000. Although the Mr 41,000 pertussis toxin substrate represents the guanine nucleotide-binding inhibitory protein Gi, the identities of the Mr 40,000 and Mr 30,000 substrates remain unclear. In any event, inhibition of the TNF-induced increase in GTPase activity and ADP-ribosylation of Gi by pertussis toxin suggested that TNF might act by increasing GTPase activity of the Gi protein. However, the results further indicate that TNF has no detectable effect on basal or prostaglandin E2-stimulated cAMP levels in HL-60 cells. Taken together, these findings indicate that a pertussis toxin-sensitive GTP-binding protein other than Gi, and possibly the Mr 40,000 substrate, is involved in the action of TNF. Finally, the demonstration that pertussis toxin inhibited TNF-induced cytotoxicity in L929 cells supports the presence of a GTP-binding protein which couples TNF-induced signaling to a biologic effect.