Proteasomal inhibition and apoptosis regulatory changes in human isolated lymphocytes: the synergistic role of dopamine.
Abnormal deposition of protein aggregates and increased susceptibility to apoptotic cell death may result from defects in the activity of the ubiquitin-proteasome system (UPS); neurotoxicity related to UPS defects seems to require dopamine to be fully expressed. The aim of this study was to investigate the pro-apoptotic effects caused by proteasomal activity inhibition, as well as the synergistic effect of dopaminergic stimulation in human lymphocytes isolated from healthy volunteers. Cells were incubated 20 h at 37 degrees C, with: (1) lactacystin, (2) increasing concentrations of dopamine or (3) mixture of dopamine and lactacystin. Activities of proteasome 20S and pro-apoptotic caspases-3 and -9 and levels of anti-apoptotic Bcl-2 were measured with fluorimetric or immunochemical assays, while a "DNA diffusion" assay was used to determine the apoptosis. Incubation of lymphocytes with lactacystin, which caused reduction of proteasomal activity, was associated with activation of caspases. A clear, dose-dependent reduction of proteasomal activity was also seen in the presence of increasing doses of dopamine, which was accompanied by a slight dose-dependent increase of caspases activities and Bcl-2 levels. Both effects on proteasome and caspase activities were enhanced when cells were simultaneously exposed to lactacystin and elevated concentrations of dopamine. Apoptosis was detected in all treated samples, but not in controls, without significant differences among the treatment groups; however, the association of dopamine and lactacystin induced a clear reduction in the number of cells being analyzed, pointing to marked cytotoxicity. Our data confirm the potentiation of cytotoxicity related to proteasome inhibition, in the presence of dopaminergic stimulation.
Bazzini
Eleonora
E
Laboratory of Functional Neurochemistry, Neurological Institute C. Mondino, Pavia, Italy.
Samuele
Alberta
A
Granelli
Marcella
M
Levandis
Giovanna
G
Armentero
Marie-Therese
MT
Nappi
Giuseppe
G
Blandini
Fabio
F
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D053453:Caspase 9 / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000633:toxicity*; D004298:Dopamine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D019254:Genes, bcl-2 / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D018655:Lymphocyte Count; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000378:metabolism; D051028:bcl-2-Associated X Protein
D015853:Cysteine Proteinase Inhibitors; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D051028:bcl-2-Associated X Protein; C067713:lactacystin; D053148:Caspase 3; D053453:Caspase 9; D046988:Proteasome Endopeptidase Complex; D004298:Dopamine; D000111:Acetylcysteine