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.

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.

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.

The metabolism-dependent maintenance of cell volume and ultrastructure in slices of Morris hepatoma 3924A.

The changes in water and electrolyte content of slices of Morris hepatoma 3924A induced by various conditions of incubation have been compared with the ultrastructural appearance of the tissue. Incubation at 1 degrees led to an increase of water, Na+, and Cl- content and to a loss of K+. There was simultaneous increase in size of the cells and intercellular spaces, loss of junctional complexes, increase in the number of microvilli, and fragmentation and dilation of the cytocavitary network. Subsequent incubation at 38 degrees in oxygenated medium led to a substantial reversal of all of these changes of composition and structure, which was well advanced within 10 min and largely complete by 60 min. The presence of 20 mM glucose in the medium somewhat enhanced the degree of recovery. A reduction of cell volume and intercellular spaces was evident both from the electron microscopic observations and measurements of the volume of inulin distribution. The presence of ouabain inhibited the net accumulation of K+ and much of the Na+ extrusion, but permitted about 50% of the net extrusion of water (accompanied by Na+ and Cl-) and had little effect on the ultrastructural recovery. The presence of glucose increased the resistance of volume and structural recovery of ouabain without releasing the inhibition of K+ accumulation. A marked feature of the recovering tissues was the Golgi apparatus, which assumed an appearance suggestive of increased activity when water extrusion was active. In slices using only endogenous substrate, cyanide and (to a lesser extent) oligomycin greatly inhibited the recovery of volume and structure. The presence of glucose permitted some recovery in the presence of cyanide. The control of cell volume in hepatoma 3924A appears to involve two separate components of water transport, one of which is sensitive, and one insensitive to ouabain. The ouabain-insensitive component appears to be especially related to the recovery of cell ultrastructure after incubation at 1 degrees, to be more sensitive to paucity of adenosine 5'-triphosphate, and to proceed by secretion of water, Na+, and Cl- into vesicles that fuse with the Golgi apparatus. This mechanism may be related to that for bile secretion in normal liver. The ouabain-sensitive component of water transport is a function of the mechanism for the coupled transport of Na+ and K+.

Transport of pyruvate in mitochondria from different tumor cells.

A comparative study of the transport of pyruvate in mitochondria isolated from normal rat liver and from three tumors has been carried out. The Km for net pyruvate uptake in mitochondria isolated from Ehrlich ascites tumor cells is practically equal to that measured in normal rat liver mitochondria while, on the other hand, it is higher in Morris hepatomas 44 and 3924A. The Vmax of pyruvate uptake is depressed in all three types of tumor mitochondria as compared to that in the rat liver mitochondria, with the depression being higher in Morris hepatoma 3924A mitochondria. The lower activity of pyruvate translocator in mitochondria isolated from tumor cells as compared to that in rat liver mitochondria is also shown by depression of the rate of pyruvate-supported oxygen uptake. The results document a decreased activity of the pyruvate translocator in tumor mitochondria which seems to be correlated with the growth rate of the tumor cells.

Calcium transport and translocation of adenine nucleotides in mitochondria from Morris hepatoma 3924A.

The interaction of Ca2+ with Morris hepatoma 3924A mitochondria and its effect on the adenine nucleotide translocation have been studied. The characteristics of the Ca2+ transport process in mitochondria from Morris hepatoma are not significantly different from those of normal liver mitochondria. The Km for Ca2+ is 2 to 3 microM, and the rate versus concentration curve exhibits hyperbolic kinetics. A lower activity of the adenine nucleotide translocation was found, probably due to the high endogenous Ca2+ content of Morris hepatoma mitochondria (123 +/- 15 nmol Ca2+ per mg protein). No further inhibition of the translocase activity was observed after isolated mitochondria had accumulated extra amounts of Ca2+. The total amount of adenine nucleotides in tumor mitochondria is one-half those present in control liver, and a significantly lower percentage of the pool is present as adenosine 5'-monophosphate.

Proton-cation translocation in tumor cell mitochondria.

The capacity of mitochondria isolated from tumor cells to conserve the transmembrane electrochemical proton gradient set up by respiration has been studied. In a K+ medium, mitochondria from Ehrlich ascites tumor cells exhibit a capacity to conserve aerobic delta microH comparable to that displayed by normal rat liver mitochondria. Mitochondria from Morris hepatoma 3924A show a decreased capacity to store delta microH+, which is principally due to lowering of delta pH. In a Na+ medium, both species of tumor mitochondria show a significant decrease of aerobic delta pH, while delta psi is the same, with respect to rat liver mitochondria. Experiments on passive swelling show that mitochondria from ascites tumor cells have an enhanced permeability to chloride salts of monovalent cations and increased activity of the Na+ (K+)-H+ exchange system of the mitochondrial membrane with respect to normal mitochondria. The enhanced activity of this system in ascites cells is also shown by the characteristics of respiration-linked proton translocation in submitochondrial particles and subsequent anaerobic proton diffusion. It is concluded that the decreased capacity of mitochondria from tumor cells to conserve aerobic delta pH is due to enhanced cyclic flow of Na+ across the membrane.

Interaction of Na+ and K+ transport with aerobic energy metabolism in slices of Morris hepatoma 3924A.

Addition of increasing concentrations of glucose to slices of Morris hepatoma 3924A greatly stimulated aerobic lactate production and reduced respiration by 20%. Neither the adenine nucleotide content of the slices nor the calculated rate of adenosine 5'-triphosphate synthesis was altered. Ouabain reduced the rate of O2 uptake (by 20 to 25%) and of aerobic lactate production (by 25 to 50%) without affecting adenine nucleotide contents. The reduction by ouabain of the calculated rate of adenosine 5'-triphosphate synthesis was similar whether the slices were utilizing only endogenous substrate or exogenous glucose also. Raising the medium K+ concentration (and correspondingly reducing Na+) partially overcame the inhibition of ion transport by ouabain and partially restored the rates of respiration and aerobic lactate production toward control levels. Electron microscopic observations of mitochondria within the slices incubated under different conditions showed variations in configuration between "orthodox," "condensed" and degenerating forms. Slices preincubated at 1 degrees showed mitochondria in the condensed form: they were restored to the orthodox configuration during incubation at 38 degrees in oxygenated medium. Oligomycin and glucose enhanced the transition, but ouabain reduced the number of mitochondria undergoing the change. The results suggest that in hepatoma 3924A utilization of adenosine 5'-triphosphate by ion transport exerts a simultaneous control of both respiration and aerobic glycolysis, which is presumably mediated by alterations in the availability of adenosine 5-diphosphate. The mitochondria undergo conformational transitions under conditions likely to affect local availability of adenosine 5'-diphosphate within cell compartments, but the transitions are not all externally added adenosine diphosphate on isolated mitochondria.

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.

Growth-related lipid peroxidation in tumour microsomal membranes and mitochondria.

Microsomes and mitochondria isolated from Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and Ehrlich ascites tumour cells exhibit a NADPH-dependent peroxidation of endogenous lipids lower than that of the corresponding fractions from rat liver. Moreover, the O2- and ascorbate-dependent lipid peroxidations are decreased in microsomes from the two Morris hepatomas. The peroxidative activity appears to be inversely related to the growth rate of the tumours. It is suggested that the low susceptibility of tumour membranes to peroxidative agents may be a factor responsible for the high mitotic activity of this tissue.

Evidence for the occurrence of the malate-citrate shuttle in intact Ehrlich ascites tumor cells.

A possible activity of the malate-citrate shuttle has been investigated in Ehrlich ascites cells by testing the effects of 1,2,3-benzenetricarboxylic acid, an inhibitor of the malate-citrate exchange, and (-)-hydroxycitrate, an inhibitor of the citrate cleavage enzyme, on the glucose-dependent oxidation-reduction rates of pyridine nucleotides and cytochrome b as well as two inhibitors glycolyzing cells. Moreover, to quantitate such an activity, the effects of these two inhibitors have been compared with those induced under the same experimental conditions by aminooxyacetate, an inhibitor of the malate-aspartate shuttle which is known to operate in this strain of ascites tumor. Both benzenetricarboxylic acid and hydroxycitrate are able to increase the reduction of pyridine nucleotides, which follows glucose addition to whole cells, to about the same extent. A much more pronounced effect is elicited by aminooxyacetate under the same condition. When n-butylmalonate is added to slow down the flux of glycolytic reducing equivalents to the respiratory chain via the malate-aspartate shuttle, benzenetricarboxylic acid or hydroxycitrate promotes an ATP-driven reversal of electron transfer. Indeed, the glucose induced reduction of cytochrome b becomes sensitive to oligomycin and the ATP level is raised significantly with respect to the value of uninhibited cells. It is concluded that the malate-citrate shuttle operates in Ehrlich ascites cells, although with a substantially lower activity with respect to the malate-aspartate shuttle.

Cytochrome P-450 deficiency and resistance to t-butyl hydroperoxide of hepatoma microsomal lipid peroxidation.

Lipid peroxidation of microsomes from rat liver and Morris hepatoma 9618A was induced by means of tert-butyl hydroperoxide (t-BuOOH). In rat liver microsomes t-BuOOH stimulated an early formation of lipid hydroperoxides (LOOH) and an increasing accumulation of malondialdehyde; t-BuOOH was completely consumed and cytochrome P-450 was rapidly destroyed. In hepatoma microsomes (60% deficiency of cytochrome P-450) a remarkable inhibition of both malondialdehyde and LOOH was observed; t-BuOOH was consumed only partially and cytochrome P-450 was destroyed slowly. In the presence of aminopyrine, malondialdehyde production was inhibited to the same extent (about 70%) in normal and tumour microsomes. The concentration of t-BuOOH required to achieve half-maximal velocity of malondialdehyde accumulation was comparable in the two microsome types. It is proposed that the deficiency of cytochrome P-450 limits the activation of t-BuOOH to the free radical species which initiate lipid peroxidation. Low cytochrome P-450 content would also affect the LOOH-dependent propagation of lipid peroxidation.

Superoxide radicals and hydrogen peroxide formation in mitochondria from normal and neoplastic tissues.

Mitochondria from beef heart, Morris hepatoma 3924A and Ehrlich ascites tumor (Lettré mutant) have been studied with respect to hydrogen peroxidase and superoxide radical formation and the presence of superoxide dismutase activity (EC 1.15.1.1, superoxide:superoxide oxidoreductase). The generation of superoxide radicals and hydrogen peroxide occurs at the level of the membrane, being present also in mitochondrial fragments. Hepatoma and ascites mitochondria have little or no superoxide dismutase activity. Superoxide radicals appear to be precursors of hydrogen peroxide formation, the reaction being catalyzed by superoxide dismutase.

Production of superoxide anions and hydrogen peroxide in Ehrlich ascites tumour cell nuclei.

Nuclei isolated from Ehrlich-Lettré ascites tumour cells catalyze the co-oxidation of epinephrine to adrenochrome in the presence of NADPH. Adrenochrome formation is sensitive to superoxide dismutase but not to scavengers of hydroxyl radicals or singlet oxygen. Addition of NADPH also initiates the production of hydrogen peroxide. Moreover measurements of superoxide dismutase activity indicate the presence of this enzyme in the ascites cell nuclei, although the sensitivity of adrenochrome formation to externally added superoxide dismutase indicates that the endogenous enzyme is not sufficient for a complete protection from superoxide radicals.

The operation of the malate-aspartate shuttle in the reoxidation of glycolytic NADH in slices of fetal rat liver.

Lactate production by liver slices from fetal rats (17th--18th day of gestation) is enhanced about two fold by aminooxyacetate, an inhibitor of aspartate transaminase (EC 2.6.1.1). Such an effect is consistent with an increase of the cytosolic NAD-redox state owing to the parallel fall in the pyruvate level, whereas the glycolytic flux does not seem to be influenced appreciably. Indeed, although the inhibitor causes a marked increase of fructose 1,6-diphosphate, glucose-6-phosphate decreases only slightly. These results suggest that in fetal rat liver the malate-aspartate shuttle is operative in the reoxidation of cytosolic NADH produced during aerobic glycolysis.

Pyruvate transport in tumour-cell mitochondria.

Tumour-cell mitochondria contain a pyruvate-transporting system exhibiting the same general properties as those described in rat liver mitochondria. The Km for net pyruvate uptake in tumour-cell mitochondria is practically similar to that measured in rat liver mitochondria but the V is lower. This difference is also shown by swelling experiments. The possible implication of these observations in the context of lactate accumulation in tumour-cell is discussed.

The control of anaerobic glycolysis by glucose transport and ouabain in slices of hepatoma 3924A.

1. The activities of glycolysis and K-+ transport have been studied in slices of Morris hepatoma 3924A incubated under anaerobic conditions in the presence of different concentrations of glucose (1-50 mM). 2. Ouabain-sensitive net transport of K-+ was observed at all glucose concentrations greater than 1 mM; ouabain reduced the rate of glycolysis by about 25% at all glucose concentrations able to support ion transport. 3. The net entry of glucose into the intracellular phase was studied at varying glucose concentrations. The rate of glucose entry was similar to the rate of glucose utilisation by anaerobic glycolysis at medium concentrations of 10 mM and less, but exceeded the rate of glycolysis at 20 mM and above. 4. The glucose entry was not Na-+-dependent and was not inhibited by ouabain. 5. The results suggest (a) that the reduction in glycolytic activity caused by ouabain is not due to an inhibition of glucose transport and (b) that the glucose transport system of this poorly differentiated hepatoma has properties similar to that of normal liver.

Superoxide dismutase content and microsomal lipid composition of tumours with different growth rates.

The content of cytosolic superoxide dismutase has been determined in Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and in ascites tumour cells (Novikoff hepatoma and Ehrlich-Lettré). The enzyme is decreased in all the tumours examined. The lowest amounts were found in the tumours with the fastest growth rates. Measurements of the lipid composition and fluidity of microsomal membranes isolated from Morris hepatomas show that also these parameters are changed in relation to the growth rate. The lipid to protein ratio and the degree of fatty acid unsaturation decrease gradually from rat liver to hepatoma 44 and 3924A microsomes. The different lipid composition is reflected also by differences in the physical environment of the bilayer, as indicated by data obtained with spin-labeled fatty acids. It is proposed that the changes in the membrane lipid composition and organization are consequent to the decrease in the protective effect of cytosolic superoxide dismutase against the O2- induced lipid peroxidation.

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.

Age-related mitochondrial lipoperoxidation in human skin.

Mitochondria were isolated from the epidermis of human subjects of different ages by a procedure that included pretreatment of the tissue with bacterial protease before homogenization. That epidermal mitochondria catalyze enzymatic lipid peroxidation in the presence of NADPH and chelated iron (ADP-Fe3+) is indicated by the production of malonaldehyde. The process does not appear to be influenced by age, and it is strongly enhanced in preparations containing a small amount of melanin.