Manganese superoxide dismutase levels are elevated in a proportion of amyotrophic lateral sclerosis patient cell lines

The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene for copper/zinc superoxide dismutase (CuZnSOD). The mechanism may involve inappropriate formation of hyroxyl radicals, peroxynitrite or malfunctioning of the SOD protein. We hypothesized that undiscovered genetic causes of sporadically occurring amyotrophic lateral sclerosis might be found in the mechanisms that create and destroy oxygen free radicals within the cell. After determining that there were no CuZnSOD mutations present, we measured superoxide production from mitochondria and manganese superoxide dismutase (MnSOD), glutathione peroxidase, NFkappaB, Bcl-2 and Bax by immunoblot. Of the ten sporadic patients we tested we found three patients with significantly increased concentrations of MnSOD. These patients also had lower levels of superoxide production from mitochondria and decreased expression of Bcl-2. No mutations were found in the cDNA sequence of either MnSOD in any of the sporadic patients. A patient with a CuZnSOD mutation (G82R) used as a positive control showed none of these abnormalities. The patients displaying the MnSOD aberrations showed no specific distinguishing features. This result suggests that the cause of ALS in a subgroup of ALS patients (30%) is genetic in origin and can be identified by these markers. The alteration in MnSOD and Bcl-2 are likely epiphenomena resulting from the primary genetic defect. It suggests also that the oxygen free radicals are part of the cause in this subgroup and that dysregulation of MnSOD or increased endogenous superoxide production might be responsible.     

Mitochondrial protein oxidation in yeast mutants lacking manganese-(MnSOD) or copper- and zinc-containing superoxide dismutase (CuZnSOD): evidence that MnSOD and CuZnSOD have both unique and overlapping functions in protecting mitochondrial proteins from oxidative damage

Saccharomyces cerevisiae expresses two forms of superoxide dismutase (SOD): MnSOD, encoded by SOD2, which is located within the mitochondrial matrix, and CuZnSOD, encoded by SOD1, which is located in both the cytosol and the mitochondrial intermembrane space. Because two different SOD enzymes are located in the mitochondrion, we examined the relative roles of each in protecting mitochondria against oxidative stress. Using protein carbonylation as a measure of oxidative stress, we have found no correlation between overall levels of respiration and the level of oxidative mitochondrial protein damage in either wild type or sod mutant strains. Moreover, mitochondrial protein carbonylation levels in sod1, sod2, and sod1sod2 mutants are not elevated in cells harvested from mid-logarithmic and early stationary phases, suggesting that neither MnSOD nor CuZnSOD is required for protecting the majority of mitochondrial proteins from oxidative damage during these early phases of growth. During late stationary phase, mitochondrial protein carbonylation increases in all strains, particularly in sod1 and sod1sod2 mutants. By using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we have found that specific proteins become carbonylated in sod1 and sod2 mutants. We identified six mitochondrial protein spots representing five unique proteins that become carbonylated in a sod1 mutant and 19 mitochondrial protein spots representing 11 unique proteins that become carbonylated in a sod2 mutant. Although some of the same proteins are carbonylated in both mutants, other proteins are not. These findings indicate that MnSOD and CuZnSOD have both unique and overlapping functions in the mitochondrion.     

Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth

We have studied the effects of overexpression of superoxide dismutase (SOD), a tumor suppressor protein that dismutes superoxide radical to H2O2, on breast cancer cell growth in vitro and xenograft growth in vivo. No previous work has directly compared the growth-suppressive effects of manganese SOD (MnSOD) and copper-zinc SOD (CuZnSOD). We hypothesized that either adenoviral MnSOD (AdMnSOD) or adenoviral CuZnSOD (AdCuZnSOD) gene therapy would suppress the growth of human breast cancer cells. After determining the antioxidant profiles of three human breast cell lines, MCF 10A, MDA-MB231, and MCF-7, we measured the effects of MnSOD or CuZnSOD overexpression on cell growth and survival in vitro and in vivo. Results demonstrated that infection with AdMnSOD or AdCuZnSOD increased the activity of the respective enzyme in all three cell lines. In vitro, overexpression of MnSOD or CuZnSOD decreased not only cell growth but also clonogenic survival in a dose- and transgene-dependent manner. In vivo, treatment of tumors with AdMnSOD or AdCuZnSOD decreased xenograft growth compared to controls. The first direct comparison of MnSOD to CuZnSOD overexpression indicated that CuZnSOD and MnSOD were similarly effective at suppressing cancer cell growth.     

Role of antioxidant enzymes in cell immortalization and transformation

Summary The role of antioxidant enzymes, particularly superoxide dismutase (SOD), in immortalization and malignant transformation is discussed. SOD (generally MnSOD) has been found to be lowered in a wide variety of tumor types when compared to an appropriate normal cell control. Levels of immunoreactive MnSOD protein and mRNA for MnSOD also appear to be lowered in tumor cells. Tumor cells have the capacity to produce superoxide radical, the substrate for SOD. This suggests that superoxide production coupled with diminished amounts of MnSOD may be a general characteristic of tumor cells. The levels of MnSOD in certain cells correlates with their degree of differentiation; non-differentiating cells, whether normal or malignant, appear to have lost the ability to undergo MnSOD induction. These observations are used to elucidate a two-step model of cancer. This model involves not only the antioxidant enzymes, but also organelle (particularly mitochondria and peroxisomes) function as a dominant theme in carcinogenesis.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

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.     

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.     

The impact of OGG1, MTH1 and MnSOD gene polymorphisms on 8-hydroxy-2′-deoxyguanosine and cellular superoxide dismutase activity in myocardial ischemia–reperfusion

Ischemia–reperfusion (I/R) injury, by inducing oxidative DNA damage, is one of the leading causes of increased patient morbidity and mortality in coronary artery by-pass grafting (CABG) surgery. 8-Hydroxyguanine (8-OHG) is an important oxidative base lesion. The 8-oxoguanine glycosylase (hOGG1) and hMTH1, which have several polymorphisms, remove 8-OHdG from the nucleotide pool. We investigated whether there are any correlations the biomarkers of oxidative stress (superoxide dismutase; SOD and 8-OHdG in serum) with genotype for two DNA repair genes (OGG1 and MTH1) and an antioxidant enzyme gene (manganese superoxide dismutase; MnSOD). Therefore, we measured DNA damage (8-hydroxy-2-deoxyguanosine; 8-OHdG) and endogenous antioxidant activity (SOD) at five different time points (T1, before anesthesia; T2, after anesthesia; T3, after ischemia; T4, after reperfusion and T5, after surgery). and also, MnSOD and MutT homolog 1 (MTH1) genes polymorphisms were genotyped by polymerase chain reaction–restricted fragment length polymorphism (PCR–RFLP) in patients undergoing coronary artery by-pass grafting (CABG) surgery. No statistically significant differences were detected in the levels of 8-OHdG and SOD in serum in terms of OGG1 Ser326Cys, MTH1 Val83Met and MnSOD Ala16Val genetic polymorphisms. Our results suggest that OGG1, MTH1 and MnSOD gene polymorphisms are not genetic risk factors for I/R injury.     

Isolation of paraquat-resistant mutants of Escherichia coli: lack of correlation between resistance and the activity of superoxide dismutase

Abstract Paraquat-resistant Escherichia coli mutants were isolated. The mutants were 10- to 50-fold more resistant to paraquat than the wild type. The wild type was more responsive to the presence of paraquat by inducing higher levels of the manganese-containing superoxide dismutase (MnSOD). Thus, in minimal medium, 0.1 mM paraquat caused a 5-fold increase in MnSOD in the wild type while it had no effect on the level of MnSOD in the mutants. Yet, 50 mM paraquat exerted a dramatic induction of SOD in the mutant strains when grown in trypticase soy yeast extract (TSY) medium. In TSY medium, catalase was not significantly affected by paraquat in all the strains tested. Resistance to paraquat in these mutant strains is, therefore, unrelated to their capacity to detoxify superoxide or hydrogen peroxide.     

Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae.

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.     

Deletions encompassing the manganese superoxide dismutase gene in the Drosophila melanogaster genome.

Two deletions, Df(2R)Sod2-11 and Df(2R)Sod2-332, are recovered that encompass the manganese superoxide dismutase (MnSOD) gene or a null mutant referred to as SOD2n283 in Drosophila. Molecular analysis has revealed that the Df(2R)Sod2-332 deletion completely uncovered both MnSOD and its adjacent gene, Arp53D, whereas Df(2R)Sod2-11 was missing the promoter region of MnSOD gene. As a consequence of reduced MnSOD expression, these deletion heterozygotes are now sensitive to oxidative stress. Complementation analysis with some recently recovered deletions in the 53C/D region has established that other essential loci exist in this interval, and second, that Arp53D function is not essential for the survival of the organism. These deletions will be instrumental in the recovery of missense substitutions in the MnSOD peptide and their influence on oxidative stress resistance.     

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.