6-hydroxydopamine-induced nuclear factor-kappa B activation in PC12 cells.

The involvement of nuclear Factor-kappa B (NF-kappa B) transcription factor in PC12 cell death triggered by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was investigated. Results show that oxidative stress generated by 6-OHDA activates NF-kappa B. When the NF-kappa B activation was inhibited by parthenolide, PC12 cell death induced by 6-OHDA was significantly increased, thus suggesting an involvement of this transcription factor in a protective mechanism against 6-OHDA toxicity. To further assess this hypothesis, we studied the involvement of NF-kappa B in the protective effect of two anti-apoptotic genes, bcl-2 and bfl-1. Although Bcl-2 and Bfl-1 expression normally protects PC12 cells from 6-OHDA, parthenolide strongly decreased the beneficial effects afforded by transgene expression. These results suggest: (1) that the transcription factor NF-kappa B is likely associated with the protection of catecholaminergic PC12 cells and (2) that the protective effects afforded by bcl-2 and bfl-1 expression may be dependent on NF-kappa activation.

Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. Although the etiology of PD is unknown, major biochemical processes such as oxidative stress and mitochondrial inhibition are largely described. However, despite these findings, the actual therapeutics are essentially symptomatical and are not able to block the degenerative process. Recent histological studies performed on brains from PD patients suggest that nigral cell death could be apoptotic. However, since post-mortem studies do not allow precise determination of the sequence of events leading to this apoptotic cell death, the molecular pathways involved in this process have been essentially studied on experimental models reproducing the human disease. These latter are created by using neurotoxic compounds such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or dopamine (DA). Extensive study of these models have shown that they mimick, in vitro and in vivo, the histological and/or the biochemical characteristics of PD and thus help to define important cellular actors of cell death presumably critical for the nigral degeneration. This review reports recent data concerning the biochemical and molecular apoptotic mechanisms underlying the experimental models of PD and correlates them to the phenomena occurring in human disease.

An enzymatic procedure for the purification of DNA restriction fragments without gel electrophoresis and ethidium bromide staining.

A rapid and simple enzymatic method for the purification of a DNA fragment from a restriction digest was developed. The method is based on the two features of exonuclease III activity: digestion of DNA from a 3'-OH at blunt or recessed ends and failure to initiate digestion at DNA ends with four-base 3' overhangs. Herein, we establish a method for purification of a DNA restriction fragment without any physical separation via gel electrophoresis. The elimination of the ethidium bromide staining and ultraviolet irradiation steps should increase the quality and the safety of the purified DNA, a matter of major concern in the perspective of human gene therapy. In addition, since the method described does not use the visualization of the restriction fragments or their difference in size it can be used to purify a DNA fragment from a pool of DNA fragments with the same size even when microquantities of material are available.

Nuclear factor-kappa B activation in permanent intraluminal focal cerebral ischemia in the rat.

Nuclear factor-kappa B (NF-kappa B) is an oxidative stress responsive transcription factor known to be activated in response to transient middle cerebral artery intraluminal occlusion. Since oxidative stress activation may largely occur during reperfusion, the aim of this study was to determine if permanent middle cerebral artery intraluminal occlusion without reperfusion induces NF-kappa B activation and the relationship of NF-kappa B activation to HSP70 expression and neuronal cell death. Our results suggest that permanent intraluminal occlusion is sufficient to induce NF-kappa B activation 7 h after the onset of occlusion. Interestingly, this activation seems to occur specifically in dying neurons of the penumbra area devoid of any HSP70 neuronal immunoreactivity. These findings are consistent with the suggested protective role of HSP70 expression and suggest that NF-kappa B activation observed in the penumbra area has a role in neuronal cell death after permanent intraluminal cerebral ischemia.

Extracellular toxicity of 6-hydroxydopamine on PC12 cells.

6-hydroxydopamine (6-OHDA) is usually thought to cross cell membrane through dopamine uptake transporters, to inhibit mitochondrial respiration and to generate intracellular reactive oxygen species. In this study, we show that the anti-oxidants catalase, glutathione and N-acetyl-cysteine are able to reverse the toxic effects of 6-OHDA. These two latter compounds considerably slow down 6-OHDA oxidation in a cell free system suggesting a direct chemical interaction with the neurotoxin. Moreover, desipramine does not protect PC12 cells and 6-OHDA is also strongly toxic towards non-catecholaminergic C6 and NIH3T3 cells. These results thus suggest that 6-OHDA toxicity on PC12 cells mainly involves an extracellular process.

A cautionary note on the use of stable transformed cells.

Gene transfection and ectopic expression is a widely used method in experimental biology. In the present report, we would like to point out that this approach may, in certain circumstances, lead to a modification of the transfected cell phenotype. Indeed, we observed that after transfection of bcl-2 gene in the neuronal PC12 cell line some of the selected clones have lost their neuronal and catecholaminergic characteristics, i.e. TH expression and ability to grow neurites in response to NGF. Thus, the resistance of some PC12-Bcl-2 clones against neurotoxic insults may not necessarily reflect the potential benefit afforded by Bcl-2 expression. We therefore encouraged authors to verify cell phenotype after stable transfection to avoid misinterpretation of their results.

Increased bax expression is associated with cell death induced by ganciclovir in a herpes thymidine kinase gene-expressing glioma cell line.

The herpes simplex virus thymidine kinase gene (HSV-tk) was stably transfected into rat C6 glioma cells (C6tk) in order to characterize the mechanisms underlying cell toxicity induced in vitro by the guanosine analog ganciclovir (GCV). The results demonstrate the efficiency of the HSV-tk/GCV system in ablating most of the tumoral cells within 7 to 8 days of treatment with 20 mivroM GCV; however, a few cells still survive. C6tk cells arrest in the S phase of the cell cycle after 2 days of drug treatment before undergoing cell death. Microscopic analysis reveals dying cells with ultrastructural characteristics consistent with apoptosis; we cannot rule out, however, that necrotic cell death may also be occurring. The cytotoxicity induced by GCV is not associated with changes in the expression of p53 protein, suggesting that cell cycle arrest and cell death may occur through a p53-independent pathway. C6tk cells constitutively express Bcl-xL and Bax proteins; when exposed to GCV, Bcl-xL levels do not change but Bax accumulation is rapidly induced. These findings suggest that the balance between Bcl-xL and Bax proteins may be of importance in determining the sensitivity of tumoral cells to GCV.

p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells.

p53, Bax and Bcl-xL proteins have been implicated in apoptotic neuronal cell death. We have investigated whether those proteins are involved in 6-OHDA-induced PC12 cell death. After a 24-h exposure to the neurotoxin (100 microM), morphological evidence for apoptosis was observed in PC12 cells. Up-regulation of p53 and Bax proteins was demonstrated 4 and 6 h, respectively, after 6-OHDA treatment; in contrast, no change in Bcl-xL levels was found. These findings suggest that p53 and Bax could be relevant markers of neuronal apoptosis as previously described in kainic acid- or ischemia-induced neuronal cell death and may participate to neuronal degeneration in Parkinson's disease.

Unlike MPP+, apoptosis induced by 6-OHDA in PC12 cells is independent of mitochondrial inhibition.

The mechanisms of 6-hydroxydopamine (6-OHDA) cytotoxicity were studied in vitro using the PC12 cell line. Following a 24 h exposure, this neurotoxin induced apoptosis and a dose-dependent decrease in cell survival. The presence of monoamine oxidase inhibitors, tranylcypromine and clorgyline, together with 6-OHDA had neither synergistic nor protective effects. Unlike 1-methyl-4-phenylpyridinium (MPP+), 6-OHDA toxicity to PC12 cells remained unchanged when glycolysis was prevented by either depleting glucose from the culture medium or growing the cells in low-glucose medium containing 2-deoxy-glucose. These results suggest that the inhibition of mitochondrial respiration is not responsible for the cell death induced by 6-OHDA.

Influence of cytokines on Toxoplasma gondii growth in human astrocytoma-derived cells.

The effects of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1-alpha (IL-1-alpha), and interleukin 6 (IL-6) on the growth of the Toxoplasma gondii RH strain were studied in vitro using a human astrocytoma-derived cell line. Cells were treated with cytokines at different concentrations at 24 h prior to infection with T. gondii tachyzoites. IFN-gamma did not induce any modification in T. gondii growth, whatever the concentration used. TNF-alpha induced a significant decrease in the total number of tachyzoites, whereas IL-1-alpha surprisingly induced an increase in the number of tachyzoites. Our results show that the effects of cytokines on T. gondii growth may be of great importance in the control of cerebral toxoplasmosis but that they can vary, depending on the cell type considered.

Met-enkephalin receptors in human gliomas.

Endogenous opioid systems (opioid peptides and receptors) are involved in many functions including the regulation of cell growth. We investigated the presence of Met-enkephalin binding sites in gliomas by displacement assays. Results demonstrated that few gliomas exhibit Met-enkephalin binding sites and that the percentage of tumours which express these binding sites strongly decreases with increasing malignancy. Moreover, we observed a shift from mu Met-enkephalin binding sites in low grade gliomas to delta Met-enkephalin binding sites in high grade gliomas. These results suggest an inactivation of the Met-enkephalinergic system in gliomas which could lead to loss of the inhibitory effect exerted by Met-enkephalin on normal astrocyte growth and thus favour progression of malignancy.

Evidence for dopamine D2 receptor mRNA expression by striatal astrocytes in culture: in situ hybridization and polymerase chain reaction studies.

The expression of dopamine D2 receptor mRNA in cultured rat striatal and cerebellar astrocytes was examined by in situ hybridization (ISH) and polymerase chain reaction (PCR). Cells double-labelled for glial fibrillary acidic protein (GFAP) immuno-histochemistry and dopamine D2 receptor mRNA (ISH) provide evidence that striatal but not cerebellar astrocytes express the dopamine D2 gene in vitro. These results were confirmed by polymerase chain reaction studies. As judged by GFAP immunostaining and morphology of the cells, this gene is almost exclusively expressed by astrocytes type 1. The expression of dopamine D2 receptor mRNA by striatal astrocytes in vitro, as found in this study, brings thus evidences for the existence of dopamine D2 receptors in such glial cells. This had been previously suggested from ligand binding studies but the typical dopaminergic nature of the binding to striatal astrocytes was left questionable. Our results with molecular biological techniques thus suggest that striatal dopamine might modulate the functions of striatal astrocytes.

Tumor necrosis factor alpha, interleukin 1 alpha, and interleukin 6 mRNA expressed by human astrocytoma cells after infection by three different strains of Toxoplasma gondii.

The role of cytokines in the pathogenesis of toxoplasmosis remains unknown to a large extent, especially in the case of reactivation that occurs in immunocompromised patients. To assess the importance of tumor necrosis factor alpha (TNF alpha), interleukin 1 alpha (IL1 alpha), and interleukin 6 (IL6), we studied the expression of these three cytokines by human astrocytoma cells after infection by three different strains of Toxoplasma gondii. The virulent RH strain, the intermediate 76K strain, and the cystogenic Prugniaud strain did not induce significantly different levels of expression of the cytokine messenger RNAs when the cytokines were studied at 1, 3, 6, and 24 h after parasitic infection. These results could indicate that infection by T. gondii strains of different virulence do not involve strong differences in TNF alpha, IL1 alpha, or IL6 expression by human astrocytoma cells.

Immunodetection of endogenous opioid peptides in human brain tumors and associated cyst fluids.

The antitumorigenic effects of endogenous opioid peptides and their presence in extracerebral tumors are well documented. In this study, methionine-enkephaline (met-enkephalin) was measured by radioimmunoassay in 108 glial and nonglial brain tumors and in 44 associated cyst fluids. By immunohistochemistry, the distribution of the peptide and its precursor, preproenkephalin A, was also analyzed. Met-enkephalin and preproenkephalin were detected in the cytoplasm and cell processes of all tumors. Moreover, for neuroectodermal tumors (i.e., gliomas, gangliogliomas, and dysembryoplastic neuroepithelial tumors), a strong inverse correlation (P < 0.0001) was observed between the met-enkephalin levels and the degree of malignancy (242.9, 148.3, 55.3, and 30.3 pg/mg protein for grade 1, 2, 3, and 4, respectively). When compared to normal tissue, this differential expression mainly results from a decrease in the opioid peptide content in high-grade neuroectodermal tumors. Meningiomas and cerebral metastases displayed low met-enkephalin levels, similar to those of grade 4 neuroectodermal tumors. Large amounts of met-enkephalin were found in all cyst fluids. These data suggest that the endogenous opioid system is an integral component of brain tumors and that met-enkephalin may represent a useful malignancy marker in neuroectodermal tumors.

Identification and characterization of an anti-tyrosine kinase factor in cystic gliomas.

In view of the frequent activation of the epidermal growth factor receptor (EGF-R) in gliomas and autocrine hypothesis, we searched for 'EGF-like' factor(s) in cystic fluids (CFs) associated with gliomas. Membranes of A431 cells, which overexpress EGF-R, were used to explore such activity in 20 CFs. In all cases CFs induced inhibition of EGF-R phosphorylation. Biochemical analysis revealed an anti-tyrosine kinase activity which was identified as a 18 kDa proteic factor. Effectiveness at high dilution and anti-proliferative effect on living cells in culture suggest that this factor may be involved in the negative regulation of glial oncogenesis.

Physical mapping of an Xq-proximal interstitial duplication in a male.

A viable duplication in the proximal long arm of the X chromosome in a boy with a malformative syndrome was delineated with molecular biology techniques using 14 probes from the X cen-Xq21 region. This analysis allowed us to refine the physical map of the X cen-Xq13 region.

[The EGF receptor pathway in human cerebral tumors]. / Etude de la voie du récepteur a l'E.G.F. dans les tumeurs cérébrales humaines.

The epidermal growth factor receptor gene is the most frequently involved proto-oncogene in human glial brain tumors, in the present series in agreement with previous reports in literature. It is therefore important to study this gene from DNA to the protein product. The vicinity of cystic fluid (C.F.) to tumor cells of the cystic wall has suggested investigation of possible "E.G.F.-like" autocrine activities in C.F. In 40% of gliomas, E.G.F.-R. gene is amplified and overexpressed. This is never observed in low grade astrocytomas. In 12% of the cases, mutations of the E.G.F.-R. gene are observed. In correlation with genomic abnormalities, E.G.F.-R. is immunoprecipitated in 40% gliomas. The basal phosphorylation of the receptor is increased in 50% gliomas. In C.F., unexpectedly, E.G.F.-R. phosphorylation inhibitory effect is observed. Its biochemical analysis suggests an anti-tyrosine kinase activity. The observation of anti-tyrosine kinase activity in C.Fs suggests the presence of negative modulatory factors of the proto-oncogene activation in tumor tissues. This could have therapeutical interest.

Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using beta-D-xyloside.

In 7-day chick embryo dorsal root ganglia and epidermis cocultures, nerve fibers avoid the epidermis. Previous studies have indicated that glycoproteic factors, secreted by epidermis, could be involved in this phenomenon. Treatment of epidermis by beta-D-xyloside, a specific proteoglycan synthesis inhibitor, abolishes the avoidance reaction. The same result is obtained when anti-chondroitin sulfate antibodies are added to the culture medium. Using HPLC and 35SO4 labeling combined with chondroitinase and hyaluronidase treatment, it has been demonstrated that chondroitin sulfate is present in the epidermal conditioned medium. This suggests that a chondroitin sulfate proteoglycan secreted by the epidermis is implicated in the neurite avoidance reaction and that epidermis could therefore control its own "noninnervation". In vivo, inhibitory influences by local extracellular components may control the guidance of growth cones during nerve pattern formation.

Effects of tunicamycin on the avoidance reaction of epidermis by sensory neurites in co-cultures.

In 7-day chick embryo dorsal root ganglia and epidermis or dermis co-cultures, nerve fibres establish contacts with dermis while avoiding epidermis. Previous results have indicated that factor(s) secreted by epidermis could be involved in this avoidance reaction. The present study demonstrates that the avoidance reaction is abolished when epidermal cells are treated by the N-linked glycoproteins synthesis inhibitor, tunicamycin. The same result is obtained after monensin treatment. The epidermal cell viability, development and total protein secretion are not significantly affected by tunicamycin, as demonstrated by trypan blue exclusion, electron microscopy and SDS-PAGE electrophoresis after 35S-methionine labelling. It has thus been concluded that the avoidance factor is glycoproteic in nature. It is also suggested that this factor possibly contains chondroitin-6-sulphate moieties.

Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro.

The neuritic growth patterns obtained on substrates made of several glycosaminoglycans (GAGs) bound to type I collagen were analysed and compared in primary cultures of chick embryo dorsal root ganglion grown in serum-free supplemented medium. In 2-day cultures grown on type I collagen or heparan sulphate (HS)-collagen surfaces, ganglionic explants exhibit a dense, symmetrical network of long, parallel neuritic processes and very few flat migrating non-neuronal cells. In contrast, on either dermatan sulphate (DS), chondroitin-6-sulphate (C6S) or hyaluronic acid (HA)-bound collagen substrates, neurons form irregular nerve fibre patterns; indeed, neurites follow convoluted paths and often, after abrupt turns, totally reverse their direction of extension. Experiments were carried out in which a choice was given to growing neural processes between collagen or GAG-collagen substrates. While growth cones elongating over type I collagen easily cross the border with HS-bound collagen surface and indiscriminately extend on this substrate, in contrast, neurites generally avoid surfaces coated with DS, C6S or HA and change their direction of growth in order to stay on collagen. The binding of DS, C6S or HA, but not HS, to type I collagen thus decreases its ability to promote neurite elongation. The interaction of neuronal cells with these extracellular matrix components by restricting neurites in their paths of extension may, therefore, play a role in the patterning of the nervous circuitry.