DOPAMINE-INDUCED APOPTOSIS IN HUMAN NEURONAL CELLS - INHIBITION BY NUCLEIC-ACIDS ANTISENSE TO THE DOPAMINE TRANSPORTER

Human neuroblastoma NMB cells take up [H-3]dopamine in a selective man ner indicating that dopamine transporters are responsible for this upt ake. These cells were therefore used as a model to study dopamine neur otoxicity, and to elucidate the role of dopamine transporters in contr olling cell death. Treatment with 0.05-0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinka ge, apoptosis-like atrophy, accumulation of apoptotic particles, DNA f ragmentation and cell death. Cycloheximide inhibited dopamine's effect , suggesting that induction of apoptosis by dopamine was dependent upo n protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, wa s blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to in hibit dopamine transport and toxicity in a drug-lice and highly select ive way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector express ing the entire rat dopamine transporter complementary DNA in the;antis ense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [H-3]dopamine uptake in a time- and dose-d ependent manner. Likewise, transient transfection of NMB cells with th e plasmid expressing dopamine transporter complementary DNA in the ant isense orientation Partially blocked [H-3]dopamine uptake. Antisense p hosphorothioate oligonucleotide 3 also decreased, dose-dependently, th e toxic effect of dopamine and 6-hydroxydopamine. Western blot analysi s with newly prepared anti-human dopamine transporter antibodies showe d that antisense phosphorothioate oligonucleotide 3 decreased the tran sporter protein level. These studies contribute to better understand t he mechanism of dopamine-induced apoptosis and neurotoxicity. Copyrigh t (C) 1996 IBRO.