Induction of adhesion/differentiation of human neuroblastoma cells by tumour necrosis factor-alpha requires the expression of an inducible nitric oxide synthase.
Neuroblastoma cell lines (SK-N-SH and SK-N-MC) were induced to differentiate, as detected by the expression of neurofilament proteins of 68 and 200 kDa, and to express adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule) after stimulation with tumour necrosis factor-alpha (TNF-alpha). This induction was accompanied by the arrest of cell growth. The induction of neuroblastoma adhesion by TNF-alpha could be inhibited by the nitric oxide synthase inhibitors, L-N-monomethyl arginine (L-NMMA) and L-N6-(1-imidoethyl)-lysine (highly specific for the inducible enzyme), but not by the inactive enantiomer D-NMMA. These results indicate that TNF-alpha induces the adhesion of neuroblastoma cells via nitric oxide. This was confirmed by the finding that the adhesion/differentiation of SK-N-SH and SK-N-MC cells can be directly induced by the addition of nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine, into the culture medium. The isoform of the nitric oxide synthase induced in human neuroblastoma cells by TNF-alpha treatment was identified enzymatically as isoform II by Western blotting and by the polymerase chain reaction. Thus TNF-alpha induces the in vitro adhesion/differentiation of human neuroblastoma cells through nitric oxide synthesized by a calcium-independent inducible form of nitric oxide synthase, clearly indicating that isoform II of nitric oxide synthase can be expressed in human neuronal cell types.
Obregón
E
E
Department of Immunology, Hospital Universitario General Gregorio Marañon, Madrid, Spain.
Punzón
M C
MC
González-Nicolás
J
J
Fernandez-Cruz
E
E
Fresno
M
M
Muñoz-Fernández
M A
MA
D000818:Animals; D001932:Brain Neoplasms / Q000201:enzymology / Q000473:pathology*; D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D051379:Mice; D019006:Neural Cell Adhesion Molecules / Q000096:biosynthesis; D009447:Neuroblastoma / Q000201:enzymology / Q000473:pathology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019006:Neural Cell Adhesion Molecules; D014409:Tumor Necrosis Factor-alpha; D019001:Nitric Oxide Synthase; D002118:Calcium