The roles of dopamine transporter and Bcl-2 protein in the protection of CV1-P cells from 6-OHDA-induced toxicity.
6-Hydroxydopamine (6-OHDA) is widely used to produce an animal model of Parkinson's disease by selectively destroying the catecholaminergic nerve system of the substantia nigra. In our previous studies we noted that dopaminergic neuroblastoma cells (SH-SY5Y) die mostly via apoptosis after exposure to 6-OHDA (< or = 100 microM) but African green monkey fibroblast (CV1-P) cells do not succumb, although in both cell lines there were increased intracellular p53 levels. This study was designed to further investigate the mechanisms underlying the p53 elevation. To test how 6-OHDA penetrates into fibroblast cells and affects p53 levels, we investigated the presence of the dopamine transporter (DAT) in CV1-P cells. We showed by western hybridization that CV1-P cells contain the DAT. The apparent entry of 6-OHDA into fibroblasts was decreased by the DAT inhibitor, 1-(2-bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenyl-propyl)piperazine (GBR 12909). Pre-treatment with GBR 12909 decreased the elevation of intracellular ROS to the control level and thus prevented the increase of p53 levels in 6-OHDA-treated CV1-P cells. Moreover, an increase of Bcl-2, an antiapoptotic protein, was detected after 6-OHDA treatment, supporting our previous results where no increase in caspase-3 activity was detected. We suggest that Bcl-2 may block the activation of the caspase cascade and protect CV1-P cells from apoptosis.
Manáková
Sárka
S
Department of Pharmacology and Toxicology, University of Kuopio, Harjulantie 1 A, PO Box 1627, FIN-70211 Kuopio, Finland. manakova@uku.fi
Puttonen
Katja A
KA
Raasmaja
Atso
A
Männistö
Pekka T
PT
D018663:Adrenergic Agents / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D016627:Oxidopamine / Q000633:toxicity*; D010879:Piperazines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018663:Adrenergic Agents; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D004338:Drug Combinations; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D016627:Oxidopamine; C043425:vanoxerine; D004298:Dopamine