Compartmentalization of the redox environment in PC-12 neuronal cells.

Neuronal redox phenomena are involved in numerous biochemical pathways and play a key role in many pathological events and clinical situations. The oxidation/reduction (redox) state present in biological compartments is a major target for possible pharmaceutical intervention and, consequently, the processes associated with its change have attracted increased attention in recent years. Here, we analyze the redox environment and its spatial compartmentalization in differentiated neuronal phenotype of PC-12 cells using a redox-sensitive protein (i.e., a mutant of the Yellow Fluorescent protein), employed ratiometrically. Redox maps of cells were generated with an elevate spatial resolution, and the spatial distributions of highly oxidized and highly reduced regions have been determined. A quantitative analysis of redox maps allows the disclosure of a peculiar spatial organization of the redox environment.

Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy.

The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.

Gene expression profiling of adrenal cortical tumors by cDNA macroarray analysis. Results of a preliminary study.

Adrenocortical carcinoma (ACC) are highly malignant tumors with poor prognosis. To verify if it is possible to assess their differential gene expression by a cDNA macroarray analysis using RNA extracted from paraffin sections, we analyzed two different cohorts of adrenal cortical adenoma (ACA) and ACC. Paraffin sections of seven ACC and seven ACA were analyzed. Transcriptional profiles were generated by commercially available c-DNA arrays testing 82 genes. Hybridization signals were quantified by densitometry and the intensity signal was compared for each gene between ACA and ACC cohorts. RNA was successfully extracted in only four out of 14 cases. Four genes displayed a significantly different expression (ACC/ACA ratio>1.5 or<0.6). Heat shock protein 60 (HSP-60) (ratio>2), Ciclin D1 and topoisomerase I (ratio>1.5) were overexpressed in the ACC cohort, while jun proto-oncogene was down-regulated. cDNA macroarray analysis from paraffin sections of adrenal tumors is feasible, despite with a low success rate. The different expression of HSP-60, Ciclin D1, jun proto-oncogene and topoisomerase I indicates that these genes may play a role in ACC pathogenesis and could represent potential diagnostic/prognostic/therapeutic target markers. Larger series of patients are necessary to confirm the biologic, diagnostic, prognostic and therapeutic implications of these findings.

Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS).

Overexpression of the c-Met/hepatocyte growth factor receptor(HGF-R) proto-oncogene and abnormal generation of intracellular oxygen species (reactive oxygen species (ROS)) have been linked, by independent lines of evidence, to cell transformation and to malignant growth. By comparing two subpopulations of the B16 mouse melanoma (B16-F0 and B16-F10) endowed with different lung metastasis capacities (low and high, respectively) we found that both the expression/phosphorylation of c-Met and the steady-state levels of ROS positively correlated with metastatic growth. shRNA-mediated downregulation of c-Met in F10 cells led to a parallel decrease in the generation of oxygen species and in metastatic capacity, suggesting that oxidants may mediate the pro-metastatic activity of the HGF receptor. c-Met activation by a ligand elicits the formation of oxidant species through the oxidase-coupled small GTPase Rac-1, a relevant downstream target of the HGF-R. Moreover, cell treatment with the catalytic ROS scavengers EUK-134 and EUK-189 attenuates Met signaling to ERKs and inhibits the anchorage-independent growth of F10 cells, consistent with a critical role for oxygen species in HGF signaling and in aggressive cell behavior. Finally, genetic manipulation of the Rac-ROS cascade at different levels demonstrated its crucial role in the pro-metastatic activity of c-Met in vivo. Thus, we have outlined a novel cascade triggered by c-Met and mediated by ROS, linked to metastasis and potentially targetable by new antimetastatic, redox-based therapies.

Protective role of MnSOD and redox regulation of neuronal cell survival.

Reactive oxygen species (ROS) play a central role in neuronal pathophysiology and in neurodegenerative disorders. However, recent evidence indicates that these molecules also operate as signaling intermediates in a variety of physiological settings, including cell protection from apoptosis. Data presented here strongly support such a dual role for oxidants in neuronal cell homeostasis. In rat pheocromocytoma cells, cell rescue by the nerve growth factor (NGF) is accompanied by a transient burst of ROS generated in the cytosol by a GTPase-dependent mechanism. Within the NGF signaling cascade, ROS lie upstream and are necessary for activation/phosphorylation of AKT/PKB and of the antiapoptotic transcription factor cAMP-responsive element-binding protein (CREB). Conversely, an increase in mitochondrial oxygen species heralds apoptosis of serum-deprived cells, and these events can be prevented by cell exposure to NGF or by treatment with the mitochondrially targeted antioxidant MitoQ. Importantly, NGF-mediated decrease of mitochondrial ROS is dependent on the transcriptional up-regulation of the manganese superoxide dismutase (MnSOD) by active CREB. These observations therefore outline a circuitry whereby cytosolic redox signaling promotes neuronal cell survival by increasing the mitochondrial antioxidant defenses.

Increased expression of cyclin E is associated with an increased resistance to doxorubicin in rat fibroblasts.

Cell cycle progression in eukaryotic cells is regulated by a family of cyclin-dependent kinases (CDKs). Cyclin E is a regulatory subunit of CDK2 and drives cells from G1 to S phase. Increased expression of cyclin E is a frequent event in human malignancies and has been associated with poor prognosis in various cancers. In this study, we evaluated the effects of cyclin E-overexpression on the sensitivity of rat fibroblasts to anticancer drugs. Cyclin E-overexpressing cells were less sensitive to doxorubicin-induced inhibition of cell growth but not to other antineoplastic drugs, such as paclitaxel, vincristine, etoposide and methotrexate. Cyclin E-overexpressing fibroblasts also displayed a reduction in ROS levels and a significantly lower increase following doxorubicin treatment compared with vector control cells. The expression of manganese superoxide dismutase (MnSOD) and its activity were increased (about 1.3-fold) in cyclin E-overexpressing derivatives compared with control cells. These results suggest that cyclin E overexpression might reduce tumour cells sensitivity to doxorubicin by affecting the expression of MnSOD and that determination of cyclin E expression levels might help to select patients to be treated with an anthracycline-based antineoplastic therapy.

The level of manganese superoxide dismutase content is an independent prognostic factor for glioblastoma. Biological mechanisms and clinical implications.

We address the issue of the role of manganese superoxide dismutase in tumorigenesis by studying a relatively homogeneous group of tumours for the correlation between amount of this anti-oxidant enzyme and prognosis. The clinical outcome of 30 patients affected by glioblastomas whose manganese superoxide dismutase content had been established at the time of first diagnosis is compared. When the survival of patients is stratified according to manganese superoxide dismutase level in the tumour, a link of these levels and prognosis can be observed. Patients with high levels of manganese superoxide dismutase show a median survival time of 6.11 months, while patients whose tumours display a low amount of MnSOD have a median survival time of 12.17 months. To assess the upstream mechanisms that sustain the increase in manganese superoxide dismutase content in brain neuroepithelial tumours, we also studied the expression of p53 in a series of 17 astrocytomas of various grading. In all tested astrocytomas, high manganese superoxide dismutase content is associated with cytoplasmic accumulation of p53. Thus glioblastomas can be divided into two distinct groups on the basis of their content of manganese superoxide dismutase, having 'better' or 'worse' prognosis, respectively. The use of this protein as a marker may help to define therapeutic strategies in the clinical management of glioblastoma.

Cell compartmentalization in redox signaling.

From a growing body of evidence on the role of Reactive Oxygen Species as intracellular signaling molecules, the concept starts to emerge that cell responses to redox changes are function of the intracellular site where oxidants are produced and/or meet their molecular targets. In particular, a major distinction between oxidative events in the cytosolic versus the mitochondrial compartment appears to exist in terms of physiological stimuli, signaling mechanisms and functional consequences. Experimental data supporting this view are reviewed here, and the potential implications of this new perspective in redox signaling are discussed.

Deregulated manganese superoxide dismutase expression and resistance to oxidative injury in p53-deficient cells.

Loss of function of the tumor suppressor protein p53 represents a very frequent event in human carcinogenesis, but the molecular mechanisms linking impaired p53 activity to increased cell malignancy are still incompletely understood. p53 is normally involved in both cell cycle control and the induction of cell death and is involved in the latter mainly through the transcriptional regulation of pro- and antiapoptotic proteins. Reactive oxygen species are known to be powerful inducers of p53 activity; moreover, they play a role in the execution of p53-dependent apoptosis. Here we show that transformed mouse fibroblasts lacking p53 are significantly more resistant than wild-type (wt) controls to the cytotoxic effect of a number of pro-oxidant treatments. Interestingly, these cells also exhibit deregulated expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD), a protein known to protect cancer cells from the oxidative injury inflicted by antitumoral cytokines and anticancer drugs. MnSOD activity was also increased in liver tissue from p53-deficient mice in comparison with wt tissue. Transient transfection of wt p53 in HeLa cells led to a significant reduction in steady-state MnSOD mRNA levels and enzymatic activity, confirming that the expression of this antioxidant enzyme is negatively regulated by p53. Forced expression of MnSOD rendered HeLa cells resistant to p53-dependent cytotoxic treatments and, in cotransfection experiments, counteracted the growth-inhibitory effect of p53. Taken together, these data identify MnSOD as a potential target for tumor suppressor protein p53 and underscore the relevance of MnSOD modulation in the context of normal p53 functions because it is consistent with many reports of abnormally increased MnSOD expression in human cancers.

A redox signaling mechanism for density-dependent inhibition of cell growth.

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.

Redox regulation of lymphocyte signaling.

Compelling evidence exists that reactive oxygen species can deliver intracellular signals in mammalian cells, and elicit a broad array of physiological responses according to the cell type, the oxidative burden and the cellular compartment where radicals are generated. When applied to immune cells, these concepts gain a particular relevance, in relation to the plasticity of immune functions and the biological complexity of lymphocyte response to antigens. Here we review some recent and somehow conflicting observations on the involvement of oxygen radicals and redox balance in lymphocyte activation, and propose models for how radical species could contribute to normal and pathological immunity.

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.

Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes.

Molecular events mediating the T-lymphocyte response to lectins are still incompletely understood, although much evidence suggests that both the mitogenic and the death-promoting effects of these agents involve the biochemical cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. Reactive oxygen species (ROS) and in particular H(2)O(2) have been shown to have a role in cell response to cytokines and growth factors. Here we report that the proliferation of mouse thymocytes in response to the mitogenic lectin concanavalin A (ConA) is strongly and selectively inhibited by the intracellular ROS scavenger N-acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surface receptors. A rapid 'burst' of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the appearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intracellular oxygen species was found in thymocytes stimulated by strong cross-linking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosine phosphorylation of multiple protein species, including a 38 kDa band able to recruit the adapter protein Grb2 and corresponding to the recently identified transducer LAT (linker for activation of T-cells), a molecule involved in linking activated TCR to the production of interleukin 2 and the proliferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c-Jun N-terminal kinase 1) in response to lectins. Taken together, these results identify ROS as important modulators of the signalling cascade initiated by mitogenic lectins in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors.

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.

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.

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.

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.

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.

Manganese content and high-affinity transport in liver and hepatoma.

Morris hepatomas 3924A and 9618A have much lower endogenous contents of Mn than normal rat liver. This work studied the uptake of Mn by slices of these three tissues over a range of concentrations from 0.05 to 100 microM. The influx was assessed with 54Mn while atomic absorption measurements determined the total content. At medium Mn from 0.05 to 5 microM, entry of 54Mn in 2 min was taken as the initial rate and within this period the apparent concentration of Mn in the cell water exceeded that in the medium. Liver showed three apparently saturable uptake systems, the medium concentrations of Mn for half-maximal uptake rate being 0.075, approximately 2, and 100 microM. Hepatoma 3924A appeared to have only two systems, the half-maximal concentration for the higher affinity mechanism being, at 0.34 microM, substantially greater than that for liver. At no concentration was the uptake rate of Mn by hepatoma 3924A less than that of liver although there was some indication that Mn uptake by 9618A was somewhat less than that by the other two tissues. It is concluded that liver and hepatoma 3924A have systems for Mn uptake with affinities that enable them to be active at the plasma concentration (approximately 0.1 microM) as well as uptake systems of less affinity. However, differences in these systems between liver and hepatomas do not account for the differences in endogenous Mn content.

The role of delta-6- and delta-9-desaturase in the fatty acid metabolism of hepatomas with different growth rate.

The fatty acid composition of microsomal membranes from Morris hepatomas 9618A, slow growing, and 3924A, fast growing, confirm the higher content in oleic acid and the loss of PUFAs of the tumours with respect to controls. The specific activities of delta-9-desaturase indicate alternative metabolic pathways for the increased production of oleic acid in the two hepatomas. The delta-6-desaturase activity is much lower in tumours than in controls. However the loss of PUFAs found in tumours seems to be mostly due to a low content in linoleic acid.