Hydroxyl free radicals induce cell differentiation in SK-N-MC neuroblastoma cells.

The SK-N-MC cell line is frequently used as a model of neuronal differentiation induced by 5-bromodeoxyuridine (BrdU). In this study, the differentiation properties of this cell line were investigated under hydroxyl free radical generation, and compared to BrdU treatment. Hydroxyl free radicals were generated in the cultures by the Fenton reaction, i.e. by simultaneous addition of ADP-Fe2+ complex and H2O2. Microscopic morphological signs, as well as the acetylcholinesterase and ganglioside sialidase activities were considered as markers of neuronal differentiation of this cholinergic neuroblastoma cell line. Apart from the altered morphological appearance, the marker enzymes displayed significant increases after both types of intervention. We suggest that hydroxyl free radicals can induce in vitro cell differentiation. They apparently play a more complex role in cell physiology than simply causing oxidative damage.

The involvement of hydroxyl free radicals in differentiation of the PC-12 rat pheochromocytoma cell line.

These experiments tested the differentiation properties of the PC-12 cell line under conditions of in vitro generation of OH&z.rad; free radicals by Fenton reaction. This involves the simultaneous addition of the following reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.025 mM), final concentrations. Superoxide dismutase activity, the increase of which is considered as a marker of differentiation, catalase and glutathione peroxidase enzyme activities were investigated, which all displayed significant increases after single and repeated interventions with hydroxyl free radicals, while the cell number remained nearly at the starting-value. It is known that the differentiation takes place when the cell number has reached a plateau. These data, therefore, suggest that hydroxyl free radicals can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damages. It is interesting that the cells can maintain high levels of these enzyme activities for a relatively long time (2 or 4 days) after a very short flux of hydroxyl free radicals.

An islet-specific CD8+ T cell hybridoma generated from non-obese diabetic mice recognizes insulin as an autoantigen.

Although CD8+ T cells play a major role in beta cell destruction in insulin-dependent diabetes in the non-obese diabetic mouse, the T cell autoantigen(s) recognized by such cells remains to be identified. Therefore, an islet-reactive, CD8+ T cell line was generated from islet-infiltrating cells and hybridized by fusion with a CD8+ alphabeta TCR- BW5147 thymoma. In the presence of islets, none of the 12 CD3+ CD8+ T cell hybridomas isolated secreted IL-2/IL-4 or IFNgamma but three were islet specific, as shown by activation induced cell death. Subclone 4A7.7.15 recognized only islets expressing H-2Kd, demonstrated islet-specific inhibition of proliferation and concomitant partial arrest in the G2/M phase of the cell cycle. Further analysis using a panel of cell lines, expressing H-2Kd, and transfected with the cDNA for various putative autoantigens in type 1 diabetes showed that 4A7.7.15 recognizes insulin as an antigen.

Cytochemical studies on the fibroblast-preadipocyte relationships in cultured fibroblast cell lines.

The theoretical basis of adipogenesis has always been a matter of debate. One concept suggests that all types of adipocytes are derived from undifferentiated connective tissue cells, whereas another concept suggests that adipocytes develop from specialized cells only that are able to accumulate fat. Many conflicting data have been published with respect to the transition of fibroblasts into preadipocytes. For example, this transition has been declared as impossible for dermal and perimysial fibroblasts. The present study analysed spontaneous accumulation of fat in various types of fibroblasts from different origin (retroocular, skin, NIH/3T3, and L929). It was found that intense Oil Red O-positive triglyceride-containing droplets accumulated in practically all types of fibroblasts provided that the cells were cultured on glass surface. When the cells were cultured on plastic surfaces, lipid staining was inhibited in a variable manner: inhibition was virtually complete in skin fibroblasts, whereas in other types of fibroblasts, inhibition was only partial. It is concluded that all types of fibroblasts can accumulate fat spontaneously, and thus can be considered as preadipocytes. Therefore, interpretations of data obtained with cultures of fibroblasts with respect to adipogenesis have to be reconsidered.

On the useful role of OH&z.rad; free radicals in differentiation of cultured human fibroblasts.

The working hypothesis assuming that oxygen free radicals cannot be considered only as harmful by-products of the oxidative metabolism has been experimentally tested. Human fibroblasts were grown in culture from the following five types of tissues: (1) normal orbital fat; (2) orbital fat of patients with endocrine ophtalmopathy (EOP); (3) normal orbital muscle; (4) orbital muscle of EOP patients; (5) skin. These fibroblasts (second to 12th passages) were treated for 2x72 h with the Fenton reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.055 mM), final concentrations. This treatment caused a slowing down of the cell proliferation, induced various morphological signs of differentiation, and significantly increased (40-150%) the total superoxide dismutase (SOD) and catalase (CAT) activities of the fibroblasts. Authors suggest that the increased expression of these enzymes may play a general role in the cell differentiation mechanisms, meaning that the generation of oxygen free radicals is an essential, useful factor even during the early phases of development, and may not be taken only as a harmful process during aging.

Ruffling and locomotion: role in cell resistance to growth factor-induced proliferation.

It has long been known that the growth rate of cells in vitro can be retarded by providing substrates of restricted area. Such experiments were performed with adhesive islets, made by depositing metals onto agarose layers through templates of various sizes. Since normal cells are unable to adhere to agarose, they become confined to the metallic surface. Using such haptotactic islets, we have studied the role of membrane ruffling and cell locomotion in the resistance of AG1523 human fibroblasts to growth factor-induced mitogenesis. Cells plated on small substrates, i.e., 2,150 microns 2 in area, initially showed vigorous ruffling, which was suppressed by 8 h after plating but had resumed again by 12 h. In contrast, cells on larger-size islets showed a rapid decline and stabilization of ruffling activity. When the growth rate was measured for single cells cultured on haptotactic islets, it was found to increase linearly from areas of 4,280 microns 2 up to 425,000 microns 2. Since the area needed to saturate the growth response was approximately 50-fold larger than the area occupied by a single cell, the growth inhibition was attributed in part to an interference with locomotion. The implication that locomotion provided positive input into growth control mechanisms was subjected to a direct test by evaluating the effect of nine polypeptide growth factors on the motility of serum-starved cells. All except TGF-beta 1 stimulated movement. Finally, the mitogenic effect of growth factors was measured by [3H]thymidine incorporation and found to be proportional to motile activities, as quantitatively assayed. We conclude that locomotion suppression is a factor in AG1523 cell resistance to growth factor-induced mitogenesis.