Manganese superoxide dismutase (SOD2) inhibits radiation-induced apoptosis by stabilization of the mitochondrial membrane

To define the molecular pathways involved in radiation-induced apoptosis and the role of the mitochondria, 32D cl 3 hematopoietic cells and subclones overexpressing either the human manganese superoxide dismutase (SOD2) transgene (1F2 and 2C6) or BCL2L1 (also known as Bcl-xl) transgene (32D-Bcl-xl) were compared for their response to radiation at the subcellular level, comparing nuclear to mitochondrial localized pathways. All cell lines showed complete detectable DNA repair by 30 min after irradiation, and clearly delayed migration of BAX and active stress-activated protein (SAP) kinases MAPK1 (also known as p38) and MAPK8 (also known as JNK1) to the mitochondria at 3 h. Radioresistant clonal lines 1F2, 2C6 and 32D-Bcl-xl showed significant decreases in mitochondrial membrane permeability, cytochrome C release, caspase 3 and poly(adenosine diphosphate-ribose) polymerase (PARP) activation at 6-12 h, and in apoptosis at 24 h. Since the nuclear-to-cytoplasm events preceding the release of cytochrome C were similar in all cell lines, and increased expression of either the SOD2 or the BCL2L1 transgene provided radiation protection, we conclude that events at the level of the mitochondria are critically involved in radiation-induced apoptosis.     

Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1

We have previously characterized more than 20 proteins induced by the immunoregulatory lymphokine IFN-gamma in human fibroblasts by their m.w. and isoelectric points determined in two-dimensional gels. Some of these proteins are induced uniquely by IFN-gamma, whereas others are also induced by IFN-alpha, TNF, or IL-1. Recent technologic advances have allowed us to begin to rapidly identify proteins induced by IFN-gamma and other cytokines by sequencing the induced proteins from blots of preparative two-dimensional gels of total cell lysates. In this study, we show that the approximately 21 kDa, isoelectric point greater than 7 protein induced by IFN-gamma is manganese superoxide dismutase (Mn-SOD), a mitochondrial protective enzyme encoded by a nuclear gene. Mn-SOD is induced by IFN-gamma and also by TNF in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of Mn-SOD mRNA is a primary, rapid, and dose-dependent response to IFN-gamma. In ACHN renal carcinoma cells, Mn-SOD mRNA and protein are induced synergistically by IFN-gamma in combination with either TNF or IL-1, and the induced protein is enzymatically active. IFN-gamma and TNF together induce Mn-SOD mRNA by more than 100-fold relative to its level in untreated ACHN cells. The induction of Mn-SOD by IFN-gamma and its synergistic induction by IFN-gamma in combination with TNF and IL-1 should protect healthy cells from the toxicity of O2- during an immune response, and may provide a mechanism for selective killing of infected cells.     

SOD2-mediated adaptive responses induced by low-dose ionizing radiation via TNF signaling and amifostine

Manganese superoxide dismutase (SOD2)-mediated adaptive processes that protect against radiation-induced micronucleus formation can be induced in cells after a 2-Gy exposure by previously exposing them to either low-dose ionizing radiation (10cGy) or WR1065 (40μM), the active thiol form of amifostine. Although both adaptive processes culminate in elevated levels of SOD2 enzymatic activity, the underlying pathways differ in complexity, with the tumor necrosis factor α (TNFα) signaling pathway implicated in the low-dose radiation-induced response, but not in the thiol-induced pathway. The goal of this study was the characterization of the effects of TNFα receptors 1 and 2 (TNFR1, TNFR2) on the adaptive responses induced by low-dose irradiation or thiol exposure using micronucleus formation as an endpoint. BFS-1 wild-type cells with functional TNFR1 and 2 were exposed 24h before a 2-Gy dose of ionizing radiation to either 10cGy or a 40μM dose of WR1065. BFS2C-SH02 cells, defective in TNFR1, and BFS2C-SH22 cells, defective in both TNFR1 and TNFR2 and generated from BFS2C-SH02 cells by transfection with a murine TNFR2-targeting vector and confirmed to be TNFR2 defective by quantitative PCR, were also exposed under similar conditions for comparison. A 10-cGy dose of radiation induced a significant elevation in SOD2 activity in BFS-1 (P<0.001) and BFS2C-SH02 (P=0.005) but not BFS2C-SH22 cells (P=0.433), compared to their respective untreated controls. In contrast, WR1065 significantly induced elevations in SOD2 activity in all three cell lines (P=0.001, P=0.007, P=0.020, respectively). A significant reduction in the frequency of radiation-induced micronuclei was observed in each cell line when exposure to a 2-Gy challenge dose of radiation occurred during the period of maximal elevation in SOD2 activity. However, this adaptive effect was completely inhibited if the cells were transfected 24h before low-dose radiation or thiol exposure with SOD2 siRNA. Under the conditions tested, TNFR1 and 2 inhibition negatively affected the low-dose radiation-induced but not the thiol-induced adaptive responses observed to be mediated by elevations in SOD2 activity.     

Manganese superoxide dismutase inactivation during Fas (CD95)-mediated apoptosis in Jurkat T cells

Manganese superoxide dismutase (MnSOD, SOD2) is an essential primary antioxidant enzyme which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix. MnSOD plays a prominent role in protection against many apoptotic stimuli. Its absence may therefore impair the cellular redox balance and enhance apoptosis. Our data show that in Jurkat T cells, following oligomerization of the Fas receptor, MnSOD is selectively degraded during apoptosis. In the presence of cycloheximide, an inhibitor of protein synthesis, the rates of cell death and MnSOD degradation were accelerated. Fas-induced MnSOD cleavage was partially inhibited in the presence of the pan-caspase inhibitor, z-VAD-fmk. MnSOD in the mitochondrial fractions was cleaved in vitro by treatment with the cytosolic fraction of Fas-activated cells. Moreover, two possible cleavage sites of recombinant hMnSOD by direct interaction with recombinant caspase-3 were noted. Cellular and mitochondrial factors were found to be necessary for the interaction. These factors include intracellular mobilization of calcium. Our data indicate that inactivation of MnSOD in receptor-mediated apoptosis by caspase-specific degradation would render the mitochondria sensitive to the steady-state production of superoxide, decrease the steady-state flux of H₂O₂, expedite the loss of mitochondrial function, and potentiate apoptosis.     

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.     

Corticotropin (ACTH) induction of tumor necrosis factor alpha by monocytes

The finding that monocytes possess specific ACTH receptors led us to determine the effect of ACTH on monokine production. Here, we report that ACTH 1-39 will induce TNF-alpha from the adherent fraction of peripheral blood leukocytes. In addition, we also found that ACTH 1-39 will potentiate IFN-gamma's induction of TNF-alpha from these cells. Since previous reports showed that ACTH inhibits IFN-gamma's activation of macrophages to a cytocidal state, our data raise questions concerning the relative role of TNF-alpha in macrophage mediated cytolysis.     

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.     

Selective cell death by water-soluble Fe-porphyrins with superoxide dismutase (SOD) activity

We investigated the effect on cell death of reactive oxygen species induced by [[5,10 (or 5,15)-bis(N-methyl-4-pyridyl)-15,20 (or 10,20) diphenyl]porphinato]iron (cis-FeMPy(2)P(2)P or trans-FeMPy(2)P(2)P) with SOD activity. The SOD activities of the cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P were measured using stopped-flow kinetic analysis. The cell viability of four cell lines treated with cis-Fe-porphyrin, trans-Fe-porphyrin, mitomycin c (MMC), or cisplatin was estimated by the alamar blue exclusion assay of the modified MTT method. The amount of cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P in the Walker 256 cultured for 24 h was 4.0 and 2.6 fmolcell(-1), respectively, indicating that the plasma membrane permeability of the Fe-porphyrins depended on their structure. Cis-FeMPy(2)P(2)P selectively killed Walker 256 and H-4-II-E as cancer cells but not FR and BRL-3A as normal cells and showed a significant cytotoxicity for the cancer cells compared with trans-FeMPy(2)P(2)P, MMC and cisplatin. We believe that cis-FeMPy(2)P(2)P as an SOD mimic converts intracellular O(2)(*-) to H(2)O(2) and that H(2)O(2) or *OH causes DNA damage and induces cell death. This result suggests that for the SOD mimic, O(2)(*-) may be useful as a target molecule to induce selective cell death between cancer and normal cells and that a metalloporphyrin having SOD activity is a new class of anticancer agents.     

Manganese superoxide dismutase (SOD2/MnSOD)/catalase and SOD2/GPx1 ratios as biomarkers for tumor progression and metastasis in prostate,colon, and lung cancer

The role of manganese-dependent superoxide dismutase (SOD2/MnSOD) during tumor progression has been studied for several decades with controversial results. While SOD2 downregulation was initially associated with tumor initiation and was proposed as a tumor suppressor gene, recent studies have reported that SOD2 might favor tumor progression and dissemination. To our knowledge this is the first time that changes in SOD2 expression in three different types of tumors, i.e., prostate, lung, and colon cancer, are studied by analyzing both SOD2 mRNA and protein levels in a total of 246 patients׳ samples. In prostate samples, SOD2 protein levels were also increased, especially in middle stage tumors. In the case of colon and lung tumors both mRNA and protein SOD2 levels were increased in malignant tissues compared to those in nontumor samples. More importantly, all metastases analyzed showed increased levels of SOD2 when compared to those of normal primary tissue and healthy adjacent tissue. Together, these results suggest that a common redox imbalance in these three types of tumor occurs at intermediate stages which then might favor migration and invasion, leading to a more aggressive cancer type. Consequently, the ratios SOD2/catalase and SOD2/Gpx1 could be considered as potential markers during progression from tumor growth to metastasis.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor

Tumor necrosis factor (TNF) induces the synthesis of protein(s) that can protect cells against subsequent killing by TNF in the presence of cycloheximide. Here we demonstrate that manganous superoxide dismutase (MnSOD), a mitochondrial enzyme involved in the scavenging of superoxide radicals (O2-), is such a protein. Overexpression of MnSOD confers increased resistance to TNF plus cycloheximide on the 293 human embryonic kidney cell line. Conversely, expression of antisense MnSOD RNA renders these cells sensitive to TNF even in the absence of cycloheximide. The TNF sensitivity of the ME-180 human cervical carcinoma cell line can also be modulated through expression of sense and antisense MnSOD RNAs. These data identify MnSOD as an important determinant of cellular resistance to TNF and implicate mitochondrially generated O2- as a key component of TNF-mediated tumor cell killing.     

Matrix metalloproteinase-2 (MMP-2) expression and regulation by tumor necrosis factor alpha (TNFalpha) in the bovine corpus luteum

Angiogenesis and tissue remodeling events in the corpus luteum (CL) are mediated by matrix metalloproteinases (MMPs). We have recently reported the cloning of bovine membrane-type 1 metalloproteinase (MT1-MMP) and have shown that active MT1-MMP is correlated to MMP-2 activity in the CL during the estrous cycle. Given the important role that MMP-2 plays in neovascularization, we became interested in understanding the role of this enzyme in the CL, a system in which angiogenesis is exquisitely regulated in the course of its lifespan. The aims of the present study were to clone bovine MMP-2 cDNA, to investigate its temporal and spatial expression in three stages of CL during the estrous cycle and to study its regulation by TNFalpha, a key cytokine regulator of CL physiology. Bovine MMP-2 cDNA was isolated from a UNI-ZAP II bovine capillary endothelial cell cDNA library and sequenced. This gene encoded a protein of 662 amino acids. Luteal tissues were collected from non-lactating dairy cows on days 4, 10, and 16 of the estrous cycle. Northern and Western blotting revealed that the levels of MMP-2 mRNA (3.1 kb) and immunoreactive pro-MMP-2 protein (68 kDa) did not differ (P > 0.05) in any age of CL examined. In addition to large luteal cells, MMP2 was localized to endothelial cells in all ages of CL by immunohistochemistry. Studies using in vitro luteal cell cultures showed that MMP-2 mRNA, protein expression and activity was upregulated by TNFalpha in a dose- and time-dependent manner. The present study suggests that MMP-2 is predominantly produced by large luteal cells and endothelial cells, and that it plays an essential role in luteal remodeling and angiogenesis. These data also suggest that cytokines such as TNFalpha may modulate these processes by regulating MMP-2 expression.     

Protein stability of mitochondrial superoxide dismutase SOD2 is regulated by USP36

SOD2 is a key mitochondrial antioxidant enzyme and its perturbation leads to oxidative cell death, which results in various disorders. In this study, we identified a deubiquitinating enzyme USP36 that regulates the protein stability of SOD2. The regulatory effect of USP36 on SOD2 was initially identified by 2-DE and MALDI-TOF/MS analyses. In addition, endogenous USP36 and SOD2 were shown to interact in an immunoprecipitation assay, which was verified using the yeast two-hybrid system. Furthermore, we demonstrated that SOD2 binds with ubiquitin molecules to form polyubiquitination chains and undergoes degradation through the ubiquitin-proteasomal pathway. Finally, USP36 was shown to be a specific deubiquitinating enzyme that reduces the ubiquitination level of SOD2 and was involved in SOD2 protein stability by extending its half-life.     

A new perspective on the role of CuZn superoxide dismutase (SOD1)

The CuZn superoxide dismutase (SOD1), a member of a group of isoenzymes involved in the scavenger of superoxide anions, is a dimeric carbohydrate free protein, mainly localized in the cytosol. The reactive oxygen species (ROS) are involved in many pathophysiological events correlated with mutagenesis, cancer, degenerative processes and aging. In the first part of this mini-review the well known role of SOD1 and ROS are briefly summarized. Following, a potential novel biological action that SOD1 could exert is described, based on the recent researches demonstrating the secretion of this enzyme in many cellular lines. Moreover, the role of impaired mutant SOD1 secretion, associated with cytoplasmic toxic inclusion, which occurs in familial amyotrophic lateral sclerosis (ALS), is summarized. In addition, a depolarization-dependent release of SOD1 in pituitary GH3 cells and in rat synaptosomes through a calcium and SNARE-dependent mechanism is reported.     

A facilitated electron transfer of copper--zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode

The direct electrochemical redox reaction of bovine erythrocyte copper--zinc superoxide dismutase (Cu(2)Zn(2)SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65 +/- 3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k(s) is 1.2 +/- 0.2 s(-1) and the anodic (alpha(a)) and cathodic (alpha(c)) transfer coefficients are 0.39 +/- 0.02 and 0.61 +/- 0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu(2)Zn(2)SOD), its Cu- or Zn-free derivatives (E(2)Zn(2)SOD and Cu(2)E(2)SOD, E designates an empty site) and the SOD reconstituted from E(2)Zn(2)SOD and Cu(2+). The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.     

Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import.