Inhibition of long-term potentiation by CuZn superoxide dismutase injection in rat dentate gyrus: involvement of muscarinic M1 receptor.

Long-term potentiation (LTP) and long-term depression represent important processes that modulate synaptic transmission that carries out a key role in neural mechanisms of memory. Many studies give strong evidences on a role of the reactive oxygen species in the induction of LTP in CA1 region of hippocampal slices that was inhibited by adding the scavenger enzyme superoxide dismutase (SOD1). Previous data showed that SOD1 is secreted by many cellular lines, including neuroblastoma SK-N-BE cells through microvesicles by an ATP-dependent mechanism; moreover, it has been shown that SOD1 interacts with human neuroblastoma cell membranes increasing intracellular calcium levels via a phospholipase C-protein kinase C pathway activation. The aim of this study was to investigate the effect of intracerebral injection of SOD1 or the inactive form of enzyme (ApoSOD) on the modulation of synaptic transmission in dentate gyrus of the hippocampus in urethane anesthetized rats. The results of the present research showed that intracerebral injection of SOD1 and ApoSOD in the dentate gyrus of the rat hippocampal formation inhibits LTP induced by high-frequency stimulation of the perforant path. This result cannot be only explained by the dismutation of oxygen radical induced by SOD1 since also ApoSOD, that lacks the enzymatic activity, carries out the same inhibitory effect on LTP induction.

Differential p63 and p53 expression in human keloid fibroblasts and hypertrophic scar fibroblasts.

The p63 gene belongs to the p53 gene family and encodes for sequence-specific transcription factors. p63 has been characterized primarily in the context of epidermis where is implicated in the establishment of keratinocyte cell fate and in maintenance of epithelial self-renewal. DeltaNp63 isoform has been showed to be involved in several kinds of human tumors of epidermal origin, even nonmalignant, for the neoplastic and proliferative potential. Here, we report the differential expression and the cellular localization of the DeltaNp63 isoform in fibroblasts isolated from human keloids and hypertrophic scars compared to normal skin. Differently from hypertrophic scar, our results show that DeltaNp63 has a nuclear localization and is overexpressed only in keloid fibroblasts, suggesting an essential role of DeltaNp63 in vivo in human keloids. Consistent with our results, we hypothesize that DeltaNp63 overexpression may be oncogenic because of its ability to block the activity of p53 since p53 is underexpressed in fibroblasts from keloids.

CuZn-superoxide dismutase in human thymus: immunocytochemical localisation and secretion in thymus-derived epithelial and fibroblast cell lines.

CuZn-superoxide dismutase (SOD) plays a key role in mediating the cellular response to oxidative stress. Its expression in normal thymus was investigated by immunohistochemical techniques and CD3-, CD68- and cytokeratin-specific staining, in order to identify thymocytes, macrophages and epithelial components. Immunocytochemical studies showed an overall CuZn-SOD thymic distribution with a prevailing concentration within thymic medulla. The analysis of CuZn-SOD release by thymus-derived epithelial and fibroblast cell lines showed the ability of both cell lines to release the anti-oxidant enzyme, especially in the presence of stress conditions as represented by serum and nutrient deprivation. These data suggest that CuZn-SOD could be a relevant antioxidant paracrine molecule in human thymus.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Secretion and increase of intracellular CuZn superoxide dismutase content in human neuroblastoma SK-N-BE cells subjected to oxidative stress.

CuZn superoxide dismutase (SOD) secretion was detected in media of [35S]cysteine-labeled human neuroblastoma SK-N-BE cells precipitated with antihuman CuZn SOD antibodies. The ability of Fe2+/ascorbate oxidative stress to induce CuZn SOD in SK-N-BE cells was evaluated by Western blot analysis. The results showed that, like human hepatocarcinoma cells and human fibroblasts, SK-N-BE cells secrete CuZn SOD. In addition, the CuZn SOD concentration was higher in cells subjected to oxidative stress than in unstressed cells. The secretion of CuZn SOD and the ability of Fe2+/ascorbate to increase its protein content in SK-N-BE cells indicates that this enzyme protects the brain from damage induced by oxidative stress.

Inhibitors of Ras farnesylation revert the increased resistance to oxidative stress in K-Ras transformed NIH 3T3 cells.

Tumor resistance to oxidative stress prevents the efficacy of cancer therapy based upon a free radical-mediated mechanism. K-ras transformed NIH 3T3 cells (E32-4-2) showed, under oxidative stress, reactive oxygen species (ROS) levels 10-fold lower and lipid peroxide levels 56% lower, compared to their nontransformed counterpart. Since p21(ras) activity depends upon farnesylation, we tested the effect of the inhibitors of farnesylation lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid on susceptibility to oxidative stress in these cells. Preincubation of cells for 24 h with 10 microM lovastatin resulted in a 10-fold increase of ROS levels and a 50% increase of lipid peroxide levels measured under pro-oxidant conditions. Similarly, preincubation of cells with 100 microM (alpha-hydroxyfarnesyl) phosphonic acid for 24 h enhanced stress-induced levels of either ROS (7.5-fold) or lipid peroxides (33%). The effect of lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid is specifically due to their ability to inhibit p21(ras) activity. In fact, inhibition of p21(ras) by transfecting E32-4-2 cells with the transdominant negative mutant of H-ras (L61, S186) led, analogously to lovastatin or (alpha-hydroxyfarnesyl) phosphonic acid treatment, to a strong increase of stress-induced ROS levels. These results suggest that farnesylation inhibitors could be used as an adjuvant therapy to improve the tumoricidal effect of cancer treatment based upon free-radical production in ras-dependent tumors.