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.     

Cell killing and induction of manganous superoxide dismutase by tumor necrosis factor-alpha is mediated by lipoxygenase metabolites of arachidonic acid.

The signalling pathways utilized by tumor necrosis factor-a (TNF) to elicit its actions have been examined in TA1 adipogenic cells. A role for lipoxygenase metabolites of arachidonic acid as mediators of TNF action in the induction of c-fos has been described. In this paper we report that acute cytotoxicity elicited by TNF, in the presence of cycloheximide (CHX), also utilizes this pathway since inhibitors of lipoxygenase action fully prevent TNF/CHX killing of several cell lines. Our data reveal that TNF induction of manganous superoxide dismutase (MnSOD) is also dependent upon lipoxygenase activity. Radical scavengers such as NAC and PDTC prevent TNF/CHX-induced cell killing and reduce MnSOD induction by TNF. Therefore, cell death by TNF/CHX treatment occurs via a pathway in which lipoxygenase products directly or indirectly operate via the generation of superoxide anions.     

Induction of superoxide dismutase and cytotoxicity by manganese in human breast cancer cells.

MnCl2 induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger), N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.     

Role of reactive oxygen species in cells overexpressing manganese superoxide dismutase: mechanism for induction of radioresistance

Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide anions (O(2)(-)) into hydrogen peroxide (H(2)O(2)). We altered the intracellular status of reactive oxygen species by introducing human MnSOD cDNA into the human ovarian cancer cell line SK-OV-3. The overexpression of MnSOD inhibited cell growth and induced a concomitant increase in the level of H(2)O(2) in SK-OV-3 cells. The cells overexpressing MnSOD were more resistant to irradiation than parental cells. MnSOD overexpression shortened the G(2)-M duration in irradiated cells. Either inhibition of p38 mitogen-activated protein kinase (p38MAPK) or scavenging free radicals blocked the induction of radioresistance by MnSOD and also abolished the shortening of the G(2)-M duration with concomitant inhibition of p38MAPK phosphorylation. Irradiation increased the generation of H(2)O(2) even more in these transfectants. These results suggest that the accumulated H(2)O(2) potentiated the activation of p38MAPK after irradiation in cells overexpressing MnSOD, which led to the protection of cells from irradiation-mediated cell death through the G(2)-M checkpoint. SK-OV-3 cells had no constitutive expression of p53, and the overexpression of MnSOD and/or irradiation did not induce p53 or p21(WAF1), which causes cell cycle arrest. Thus, our results suggest that MnSOD alters the cell cycle progression of irradiated cells independently of p53 and p21(WAF1).     

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.     

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.     

Expression of superoxide dismutases, catalase, and glutathione peroxidase in glioma cells

Four primary antioxidant enzymes were measured in both human and rat glioma cells. Both manganese-containing superoxide dismutase (MnSOD) and copper-zinc-containing superoxide dismutase (CuZnSOD) activities varied greatly among the different glioma cell lines. MnSOD was generally higher in human glioma cells than in rat glioma cells and relatively higher than in other tumor types. High levels of MnSOD in human glioma cells were due to the high levels of expression of MnSOD mRNA and protein. Heterogeneous expression of MnSOD was present in individual glioma cell lines and may be due to subpopulations or cells at different differentiation stages. Less difference in CuZnSOD, catalase, or glutathione peroxide was found between human and rat glioma cells. The human glioma cell lines showed large differences in sensitivity to the glutathione modulating drugs 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO). A good correlation was found between sensitivity to BCNU and the activities of catalase in these cell lines. Only one cell line was sensitive to BSO and this line had low CuZnSOD activity.     

Protection from tumor necrosis factor-mediated cytolysis by overexpression of plasminogen activator inhibitor type-2

Pretreatment of HT-1080 fibrosarcoma cells with tumor necrosis factor (TNF) induced resistance to the cytolytic activity of this cytokine in combination with cycloheximide. This resistance correlated with the synthesis of plasminogen activator inhibitor type-2 (PAI-2). HT-1080 cells were transfected with a PAI-2 expression vector in both sense and antisense orientation. The resistance to TNF-mediated cytolysis of transfected cell clones was correlated with the level of PAI-2 expression. Cells expressing antisense PAI-2 RNA showed reduced expression of PAI-2 and increased sensitivity to TNF-mediated cytolysis. Cells expressing constitutively PAI-2 were treated with TNF and cycloheximide to select cells with increased resistance to cytolysis and enhanced PAI-2 expression. PAI-2 gradually disappeared during a treatment with TNF and cycloheximide. This finding suggested that PAI-2 formed a complex with a target proteinase, which could be involved in mediating the cytolytic activity of TNF.     

Differential effects of tumor necrosis factor and asbestos fibers on manganese superoxide dismutase induction and oxidant-induced cytotoxicity in human mesothelial cells

We compared induction of manganese superoxide dismutase (MnSOD) by asbestos fibers and tumor necrosis factor (TNF) using cultures human mesothelial cells. Transformed pleural mesothelial cells (MET 5A) were exposed for 48 h to amosite asbestos fibers (2 g/cm²), to TNF (10 Ng/ml), and to the combination of these two. TNF and amosite+TNF caused significant MnSOD mRNA upregulation. Similarly MnSOD specific activity was increased by TNF (290% increase) and the amosite+TNF combination (313% increase) but not by amosite alone. In cell injury experiments, amosite and amosite+TNF exposures caused significant cell membrane injury when assessed by lactate dehydrogenase release, which was 31% and 57% higher than in the unexposed cells. However, only the amosite+TNF combination caused significant depletion of cellular high-energy nucleotide when expressed as percentage of [¹⁴C]denine labeling in cellular high-energy nucleotides. The nucleotide levels were 91.5 ± 2.0% in the unexposed cells, 89.9 ± 3.9% in amosite-exposed cells, 90.1 ± 2.2% in TNF-exposed cells, and 79.8 ± 9.4% in amosite+TNF-exposed Amosite+TNF-exposed cells were also most sensitive to menadione (20 mol/L, 2 h), a compound which generates superoxide radicals intracellularly. In conclusion, our data suggests that in human mesothelial cells inflammatory cytokines but not asbestos fibers alone can cause MnSOD induction. In this study, however amosite asbestos+TNF treatment rendered these cells more vulnerable to oxidant-induced cell damage despite elevated MnSOD activity.Abbreviations MnSOD manganese superoxide dismutase - TNF tumor necrosis factor - LDH lactate dehydrogenase     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

Differential regulation of antioxidant enzymes in response to oxidants.

We have demonstrated the selective induction of manganese superoxide dismutase (MnSOD) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A 3-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and MnSOD were less striking. Expression of catalase, MnSOD, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc SOD (CuZnSOD) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. MnSOD mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZnSOD mRNA remained unchanged. The 3-fold induction of MnSOD mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. MnSOD mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive MnSOD protein was produced in a corresponding dose- and time-dependent manner. Induction of MnSOD gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.     

Antioxidant defense systems of two lipidopteran insect cell lines

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O(2)(*-)) to hydrogen peroxide (H(2)O(2)) and ascorbate peroxidase (APOX) for reducing the resulting H(2)O(2) to H(2)O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (approximately two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H(2)O(2) to H(2)O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H(2)O(2). Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.     

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.     

Oxygen toxicity in control and H2O2-resistant Chinese hamster fibroblast cell lines

Following exposure to 95% oxygen, clonogenic cell survival was assayed and qualitative morphologic changes were observed in a Chinese hamster fibroblast cell line (HA-1). The time in 95% O2 necessary to clonogenically inactivate 90% of the cells was inversely related to the cell density of the cultures at the beginning of hyperoxic exposure (from 1 to 6 X 10(4) cells/cm2). The O2-induced loss in clonogenicity and evidence of morphologic injury were shown to be significantly delayed (17-22 h) in an H2O2-resistant variant of the parental HA-1 cell line. After the delay in onset of clonogenic cell killing or morphologic injury, the process of injury proceeded in a similar fashion in both cell lines. The H2O2-resistant cell line demonstrated significantly greater catalase activity (20-fold), CuZn superoxide dismutase activity (2-fold), and Se-dependent glutathione peroxidase activity (1.5-fold). The greater activities of CuZn superoxide dismutase and catalase were accompanied by similarly greater quantities of immunoreactive protein as determined by immunoblotting. These data demonstrate that the cells adapted and/or selected for growth in a highly peroxidative environment also became refractory to O2-induced toxicity, which may be related to increased expression of antioxidant enzymes. However, the magnitude of this cross-resistance to O2 toxicity was less than the magnitude of the cellular resistance to the toxicity of exogenous H2O2, suggesting that in this system the toxicity of 95% oxygen is not identical to H2O2-mediated cytotoxicity.     

Induction of resistance to TNF cytotoxicity and mitochondrial superoxide dismutase on U-937 cells by 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited the cytotoxicity of tumor necrosis factor (TNF) in a human monoblastic leukemic cell line, U-937. Combination of TNF and 1,25(OH)2D3 remarkably increased mitochondrial superoxide dismutase (mSOD) of U-937 cells, TNF alone increased it only slightly and 1,25(OH)2D3 alone did not. The cytosolic SOD (cSOD) activity was not changed by TNF or/and 1,25(OH)2D3. The mSOD activity was not inhibited by 2 mM KCN, suggesting that mSOD should be a manganese SOD (MnSOD). These results suggest that 1,25(OH)2D3 may reduce the susceptibility to TNF cytotoxicity of U-937 cells by enhancing the ability of inducing MnSOD by TNF.