L-DOPA and glia-conditioned medium have additive effects on tyrosine hydroxylase expression in human catecholamine-rich neuroblastoma NB69 cells.
The aim of this study was to investigate the effect of L-DOPA and glia-conditioned medium (GCM) on cell viability, tyrosine hydroxylase (TH) expression, dopamine (DA) metabolism and glutathione (GSH) levels of NB69 cells. L-DOPA (200 microM) induced differentiation of NB69 cells of more than 4 weeks in vitro, as shown by phase-contrast microscopy and TH immunocytochemistry, and decreased replication, as shown by 5-bromodeoxyuridine immunostaining. L-DOPA did not increase the number of necrotic or apoptotic cells, as shown by morphological features, Trypan Blue, lactate dehydrogenase activity, bis-benzimide staining and TUNEL assay. Furthermore, L-DOPA (200 microM) increased Bcl-xL protein expression. Incubation of cells with L-DOPA (50, 100, 200 microM) for 24 h resulted in an increase in TH protein levels (174, 196 and 212% versus control). Neither carbidopa, an inhibitor of L-aromatic amino acid decarboxylase enzyme, nor L-buthionine sulfoximine, which inhibits GSH synthesis, or ascorbic acid, an antioxidant, blocked the L-DOPA-induced effect on TH protein expression. L-DOPA (0, 50, 100 and 200 microM) plus GCM further increased the amount of TH protein (346, 446, 472 and 424%). L-DOPA (200 microM) increased TH protein levels to 132, 191 and 245% of controls after incubation for 24, 48 and 72 h. DA metabolism in NB69 cells was increased in cultures treated with either L-DOPA (200-300 microM) or GCM and these two agents had a synergistic effect on DA metabolism. In addition, L-DOPA (200 microM) or/and GCM-treated cells increased their GSH extracellular levels (223, 257, 300% of controls) after 48 h of treatment. The L-DOPA-induced increase of TH protein expression in NB69 cells was independent of DA production, free radicals and GSH up-regulation.
Rodríguez-Martín
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Departmento Neurobiología-Investigación, Hospital Ramón y Cajal, Madrid, Spain.
Canals
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Casarejos
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de Bernardo
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Handler
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Mena
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D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002230:Carbidopa / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004298:Dopamine / Q000378:metabolism; D015259:Dopamine Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007980:Levodopa / Q000494:pharmacology*; D009447:Neuroblastoma; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein
D000975:Antioxidants; C494811:BCL2L1 protein, human; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D015259:Dopamine Agents; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D007980:Levodopa; D019328:Buthionine Sulfoximine; D014446:Tyrosine 3-Monooxygenase; D005978:Glutathione; D002230:Carbidopa; D001205:Ascorbic Acid; D004298:Dopamine