Pierre Broquet

Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to γ-radiation but activation of this sequence is defective in radioresistant SQ20B cells

Purpose : To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to γ-rays. Materials and methods : Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (ΔΨm) , reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry. Results : In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in ΔΨm and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, γ-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway. Conclusions : Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W‐7 [N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin‐dependent kinases, gave the same concentration‐dependent inhibition profile of sialyltransferase as desipramine, whereas H‐7 [1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide‐dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin‐dependent system.

A brain sialyltransferase having a narrow specificity for O-glycosyl-linked oligosaccharide chains☆

The existence of a brain sialyltransferase catalyzing the specific transfer of NeuAc on native fetuin was demonstrated. This enzyme was not able to sialylate either asialofetuin or desialylated and nondesialylated orosomucoid, transferrin, and bovine submaxillary mucin. It required the presence of Mn2+ for optimal activity. Moreover, in fetuin, this activity was closely related to the proportion of NeuAc residues, but in liver tissue sialylation occurred only onto asialofetuin. In native fetuin, sialylation took place on O-glycan chains to give an O-disialyltetrasaccharidic structure. The Gal----GalNAc----protein was not an acceptor, but alpha-NeuAc-(2----3)-Gal----GalNAc----protein was, suggesting a specific transfer alpha-(2----6) to the GalNAc residue.

Biosynthèse des glycoprotéines. XXI. Étude de la mannosyl-transférase microsomique des splénocytes

Summary An enzymatic system catalyzing mannose transfer to endogenous proteins from the coenzymatically active form of the ose, the GDP-mannose, is present in splenic cellular microsomes. This mannosyltransferase shows an optimum pH of 5.8, an optimum temperature of 30°C, and is highly sensitive to any temperature or ionic strength increase. Its Km for GDP-mannose is 0.1 μ M. The enzyme is activated by Mn++ and Mg++, by mono- and triphosphate nucleosides and inhibited by diphosphate nucleoside and X-100 Triton. Splenic microsomic mannosyl-transferase proves very sensitive to any alternation of subcellular architecture at the microsomic level.

Effect of retinoic acid on two glycosyltransferase activities in c6 cultured glioma cells

1. Activity of two glycosyltransferases was studied in retinoic acid-treated C6 cultured glioma cells. 2. The beta-galactoside alpha 2,3-sialyltransferase transferring N-acetylneuramin onto the O-glycans residues of glycoproteins was activated up to twice after chronic treatment (from 24 to 96 hr) with all-trans retinoic acid. 3. No effect was observed for shorter treatments. 4. On the opposite, the N-glycan galactosyltransferase activity remained unchanged whatever the length of retinoic acid treatment was. 5. The activatory effect was not dependent on isomery, as all-trans and 13-cis retinoic acid isomers were both activators of the C6 glioma cell sialyltransferase. 6. Measurement of adhesion of retinoic acid-treated cells using labelled plasma membranes showed an enhancement of adhesion in correlation with enhancement of sialyltransferase activity.

Study of o-glycan sialylation in c6 cultured glioma cells: Regulation of a β-galactoside α2,3 sialyltransferase activity by ca2+/calmodulin antagonists and phosphatase inhibitors

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.

Cartilage degradative enzymes in human osteoarthritis: Effect of a nonsteroidal antiinflammatory drug administered orally

The activity of stromelysin and collagenase was determined in fibrillated human OA cartilage using labeled proteoglycans and type II collagen as substrates. In vitro paracetamol had no effect on metalloprotease whereas TA induced a significant inhibition of stromelysin. In cartilage and synovium from nine patients treated with TA and nine patients treated with paracetamol during 8 weeks before surgery for hip OA, stromelysin activity was significantly lower in the TA than in the paracetamol group. The results suggest that TA has a potential chondroprotective effect in OA.

Study of O-glycan sialylation in C6 cultured glioma cells: Evidence for post-translational regulation of Aβ-galactoside α2,3 sialyltransferase activity by N-glycosylation

We have studied the Gal beta 1-3GalNAc-R alpha 2,3 sialyltransferase from C6 glioma cells transferring Neu5Ac from CMP-Neu5Ac onto O-glycans of glycoproteins. Using synchronized C6 glioma cells, we showed that the alpha 2,3 sialyltransferase activity was inhibited by tunicamycin to a greater extend than DNA and protein biosynthesis suggesting inhibition of N-glycosylation of this enzyme. Additional demonstration of N-glycosylation of the alpha 2,3 sialytransferase was provided through ConA-Sepharose binding. Treatment of partially purified alpha 2,3 sialytransferase by peptide-N-glycosidase F showed a significative inhibition demonstrating that N-glycan moiety is required for complete activity of the C6 glioma cell alpha 2,3 sialyltransferase.

Serotonin-stimulated phospholipase A2 and collagenase activation in chondrocytes from human osteoarthritic articular cartilage

We have previously described several receptors on the chondrocyte membrane. In an attempt to further characterize the coupling mechanisms of serotoninergic receptors, here we examined the involvement of serotonin in the phospholipase A2 activity. Serotonin dose‐dependently stimulated phospholipase A2. This activation enhanced collagenase type II activity and had no effect on proteoglycanase activity.

Calmodulin-dependent collagenase and proteoglycanase activities in chondrocytes from human osteoarthritic cartilage.

Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation.