Defective gene expression of MnSOD in cancer cells.

Reactive oxygen species (ROS) have been frequently implicated in the initiation and promotion phases of carcinogenesis. Antioxidant enzymes, which can antagonize this process, are lowered in a number of malignancies. The enzyme most commonly decreased is the mitochondrial Mn-containing superoxide dismutase (MnSOD) encoded by a nuclear gene mapped on the band 6q21, a region frequently deleted in several human tumours. The close association of del(6q) with diminution of MnSOD has led to suggest that MnSOD might be a new type of tumour-suppressor gene. This hypothesis is also sustained by the finding that transfection of MnSOD cDNA into human melanoma cell lines suppress the malignant phenotype. There are, however, conflicting observations that tend to ascribe the deficiency of the MnSOD activity more to a defect in the expression of the gene than to its deletion. In many transformed cell lines, including some with marked del(6q), there is no change in the dosage of the MnSOD gene and the enzyme is highly inducible by various pro-oxidant agents. Transition metals (Mn, Fe) have been found to be highly deficient in human and rodent tumours. Owing to the second messenger function of ROS in activating transcription factors (NF-kB, AP-1) and to the ability of Mn to facilitate the dismutation of O2- to H2O2 and of Fe to participate in the Fenton reaction, we propose that in the early stage of carcinogenesis an impairment of the signal transduction machinery, related to the metal deficiency, might limit the binding to DNA of transcription factors and cause the defect in the MnSOD gene expression.

Manganese deficiency and transcriptional regulation of mitochondrial superoxide dismutase in hepatomas.

The presumed involvement of the transition metals manganese and copper in the regulation of the expression of the Mn- and CuZn-containing superoxide dismutase genes has been investigated in normal and neoplastic tissues of the rat. Two hepatomas of the Morris line have been employed, the slow growing, highly differentiated 9618A and the fast growing, poorly differentiated 3924A. The data obtained indicate a control at the pretranslational level of the Mn-containing enzyme, presumably exerted by the manganese ion. The CuZn-containing superoxide dismutase is also regulated pretranslationally in the normal tissues examined and in the hepatoma 3924A. However, there is no indication for the involvement of the copper ion, which in the liver is mostly located in the cytosol bound to CuZnSOD, in such regulation. The possible role of a reduced redox state, concomitant to the manganese deficiency in hepatoma tissues, in the down regulation of Mn-containing superoxide dismutase is discussed.

Iron modulation of LPS-induced manganese superoxide dismutase gene expression in rat tissues.

Mitochondrial superoxide dismutase (MnSOD) is usually diminished in cancer cells. We observed that in vivo treatment with LPS produces a strong increase of MnSOD mRNA levels and a weak induction of an inactive protein in rat hepatocarcinomas. In normal liver iron deficiency, obtained with desferrioxamine administration, produces a decrease in the MnSOD induction by LPS, indicating that such induction could depend on tissue iron content. However, no change in MnSOD mRNA has been observed in iron-overloaded tumor tissue. Thus, iron is possibly involved in the transcriptional regulation of the protein, in combination with some other unknown factor that appears to be deficient in tumor cells.

Oxidative stress and overexpression of manganese superoxide dismutase in patients with Alzheimer's disease.

Substantial evidence supports the hypothesis that oxygen free radicals are involved in various neurodegenerative disorders. To assess the presence of oxidative stress in Alzheimer's disease (AD) we examined the activity of the enzyme copper-zinc superoxide dismutase (CuZnSOD) in red blood cells, the levels of the mitochondrial inducible enzyme manganese superoxide dismutase (MnSOD) mRNA in lymphocytes, and the total radical-trapping antioxidant capacity (TRAP) in plasma of AD patients and in a group of age-matched non-demented controls. We found that CuZnSOD activity (P < 0.01 vs. controls) was significantly increased as well as the MnSOD mRNA levels while the total antioxidant status (P < 0.001 vs. controls) was decreased in AD patients. These findings support the role of oxidative alterations in the pathogenetic mechanism underlying AD neurodegeneration.

Transcriptional regulation of MnSOD by manganese in the liver of manganese-deficient mice and during rat development.

Evidence is reported that liver manganese deficiency, whether artificially produced by the administration of a Mn-deficient diet, or physiologically occurring in the neonatal life, in mice and rats respectively, causes the down-regulation of the manganese-containing superoxide dismutase at (pre)-transcriptional level. These observations, in addition to previous data concerning Mn-deficiency and the low level of expression of MnSOD in Morris hepatomas, strongly support the role played by the metal ion in the control of the MnSOD by a mechanism of gene activation. While the molecular events taking place in such regulation are not yet identified, the involvement of reactive oxygen species (ROS) as second messengers in the activation of specific transcription factors is suggested.

Messenger RNA for manganese and copper-zinc superoxide dismutases in hepatomas: correlation with degree of differentiation.

Total and polyadenylylated RNA have been isolated from two Morris hepatomas with different degree of differentiation and from the normal liver of the corresponding tumor-bearing inbred rats. The analysis of mRNA has been performed by Northern hybridization using 32P-dA-tailed synthetic deoxyoligonucleotide probes, 33-mer for Mn superoxide dismutase (SOD) and 36-mer for CuZnSOD, derived from the nucleotide sequences of the rat enzyme cDNAs. Two distinct mRNA species (about 850 and 1080 nucleotides) have been identified by using the MnSOD probe. CuZnSOD is translated from a single message of about 720 nucleotides. The total MnSOD mRNA concentration is decreased by 43% and 57% in the hepatomas 9618A (highly differentiated) and 3924A (poorly differentiated), respectively. CuZnSOD mRNA is practically unchanged in the hepatoma 9618A whereas it is reduced by 80% in the hepatoma 3924A. Comparison of the enzyme activities and mRNA levels indicates a good correlation only for hepatoma 3924A, suggesting that the changes of both SODs are regulated pretranslationally. From the data obtained it is also inferred that the mRNA levels of MnSOD respond more readily than those of CuZnSOD to changes in differentiation.

Ethanol treatment up-regulates the expression of mitochondrial manganese superoxide dismutase in rat liver.

On the basis of the well-known effect of ethanol poisoning on the prooxidant/antioxidant balance of human and rodent liver we tested the response of the mitochondrial manganese-containing superoxide dismutase (MnSOD) in the liver of rats following an acute ethanol load or chronically intoxicated with an alcohol-supplemented solid diet for three weeks. In both conditions the enzyme activity and messenger RNA were monitored. In the acutely treated animals MnSOD was induced (post-)translationally already at 3 hours after ethanol injection, reached the maximum level (about 50% increment) at 9 hours and decreased thereafter. Chronic ethanol feeding caused an up-regulation of the enzyme at the mRNA level, with a good correlation between the transcript and the enzyme activity during the first two weeks of treatment. After 20 days the mRNA level dropped to normal, whereas the activity still remained high. Chronic alcohol intake also led to a significant decrease in the content of vitamin E in the liver mitochondrial and microsomal fractions, suggesting the occurrence of an enhanced lipid peroxidation, consequent to the ethanol-induced oxidative stress. The response of MnSOD appears to be a protective mechanism that the genetic machinery builds up to partially overcome such a condition.

Diethyldithiocarbamate treatment up regulates manganese superoxide dismutase gene expression in rat liver.

In vivo experiments demonstrate that rat liver manganese-containing superoxide dismutase (MnSOD) is up-regulated at the transcriptional level following the inactivation of copper-zinc superoxide dismutase (CuZnSOD). CuZnSOD activity was inhibited by the administration of the copper chelating agent diethyldithiocarbamate (DDC). This CuZnSOD inactivation is likely associated with an intracellular oxidative stress. Indeed the antioxidant N-acetyl-cysteine (NAC) completely prevents the MnSOD mRNA up-regulation observed after DDC administration. Evidence is also provided that an approximately 50% diminution of the total iron content in the tissue, which follows the in vivo administration of the iron chelator desferrioxamine (DESF), reduces the amount of MnSOD induction achieved by DDC treatment. Both NAC and DESF significantly down-regulate MnSOD gene expression also in normal untreated rat liver. While the observed inhibitory effect of NAC in MnSOD mRNA up-regulation can be ascribed mainly to its antioxidant property, iron chelation could act with an antioxidant effect and/or affecting some iron-dependent factor(s) possibly involved in MnSOD gene regulation. It is proposed that this metal could have a role among factors that sense and/or trigger transcription of the MnSOD gene.

Increased growth capacity of cervical-carcinoma cells over-expressing manganous superoxide dismutase.

Increases in the expression of manganese-dependent superoxide dismutase (MnSOD) have been detected in several classes of human and experimental tumors and appear to correlate with poorer prognosis in human neuro-epithelial, ovarian and cervical tumors. To delineate the relevance of MnSOD expression to tumor-cell growth and survival, a human MnSOD cDNA was over-expressed in the HeLa cervical-carcinoma cell line. MnSOD over-expression had marginal effects on the growth of HeLa cells in standard medium, but markedly protected the cells from growth suppression and cell death in conditions of serum deprivation. Serum starvation did not affect expression of endogenous MnSOD in wild-type HeLa cells, but was associated with increases in cell death and in the generation of intracellular oxygen radicals. By contrast, in HT29 colon-carcinoma cells, which are relatively resistant to growth-factor withdrawal, serum deprivation was associated with increases in MnSOD expression and activity. Together these observations suggest that MnSOD provides a mechanism for counteracting the intracellular oxidative processes that impair cell growth and viability in the context of growth-factor withdrawal and, in this context, may promote tumor-cell survival in vivo in conditions normally unfavorable to cell growth.

The level of MnSOD is directly correlated with grade of brain tumours of neuroepithelial origin.

The oxy-radical scavenger enzyme manganese superoxide dismutase (MnSOD) may act in the capacity of a tumour-suppressor gene. To address the issue of its role in tumour transformation and progression in vivo, we evaluated the content of this enzyme in 33 brain tumours of neuroepithelial origin with different degrees of differentiation (WHO grade II-IV) by means of Western blot and immunohistology. Our results show that immunoreactive MnSOD increases in a direct relationship with tumour grade and is therefore inversely correlated with differentiation. The increase in induced at a pretranscriptional level and is apparently specific to brain tumours of neuroepithelial origin. Approximately 30% of grade IV tumours display low levels of MnSOD content, and preoperative radiotherapy and brachytherapy result in low amounts of enzyme. Based upon these observations, we suggest that MnSOD cannot be considered a classical tumour-suppressor gene.

Regulation of manganese superoxide dismutase (MnSOD) in chronic experimental alcoholism: effects of vitamin E-supplemented and -deficient diets.

In order to investigate the pathogenic mechanism responsible for liver injury associated with chronic alcoholism, we studied the effects of different dietary vitamin E levels in chronically ethanol (EtOH)-fed rats on the activity and mRNA regulation of the manganese superoxide dismutase (MnSOD) enzyme. Evidence is accumulating that intermediates of oxygen reduction may in fact be associated with the development of alcoholic liver disease. Since low vitamin E liver content seems to potentiate EtOH-linked oxidative stress, we studied the effect of EtOH treatment in livers from rats fed a diet deficient or supplemented with vitamin E. Chronic EtOH feeding enhanced hepatic consumption of vitamin E in both groups of EtOH-treated animals, irrespectively of the vitamin E level of the basal diet and the effect was observed in both the microsomal and mitochondrial fractions. Both EtOH-fed groups exhibited increased MnSOD gene expression, while the enzyme activity was enhanced only in the vitamin E-deprived group of EtOH-treated animals. The significant increase in manganese liver content found only in this last group could explain the rise of enzyme activity. In fact, in the absence of a parallel increase of the prosthetic ion manganese, MnSOD mRNA induction was not accompanied by a higher enzymatic activity. These findings support the role of oxidative alteration in the EtOH-induced chronic hepatotoxicity in which MnSOD response might represent a primary defence mechanism against the damaging effect of oxygen radical species.