Down-regulation of Bcl-2, activation of caspases, and involvement of reactive oxygen species in 6-hydroxydopamine-induced thymocyte apoptosis.
OBJECTIVE
Our previous work showed that 6-hydroxydopamine (6-OHDA) induced mouse thymocytes to undergo apoptosis both in vivo and in vitro. In the present study, we further investigated the mechanisms of 6-OHDA-induced apoptosis in vitro.


METHODS
Naive mouse thymocytes were cultured with 6-OHDA. The percentages of apoptotic cells were quantified by propidium iodide staining, and DNA fragmentation was detected by agarose gel electrophoresis. Intracellular Bcl-2 was analyzed by immunofluorescence staining. Cu/Zn superoxide dismutase (Cu/Zn-SOD) activities were measured by the SOD-525 method.


RESULTS
The apoptotic effect of 6-OHDA was blocked by desipramine, a catecholamine uptake blocker. Treatment with 6-OHDA caused a reduction in Bcl-2 expression. VAD-FMK, a broad-spectrum caspase inhibitor, and DEVD-CHO, a potent inhibitor of caspase-3, could block 6-OHDA-induced thymocyte apoptosis. However, the specific caspase-1 (ICE) inhibitor YVAD-CMK had no effect. This cell death process was prevented by the protein synthesis inhibitor cycloheximide and by antioxidants. The level of Cu/Zn-SOD activities also decreased after cells were exposed to 6-OHDA.


CONCLUSIONS
These results suggest an apoptotic effect of 6-OHDA via the uptake of this neurotoxin by thymocytes, and that down-regulation of Bcl-2, activation of caspases, such as caspase-3 but not caspase-1, generation of reactive oxygen species, and new synthesis of proteins are involved in this process.
Tsao
Chiung-Wen
CW
Department of Nursing, Chung Hwa College of Medical Technology, Tainan Hsien, Tainan, Taiwan, ROC.
Cheng
Juei-Tang
JT
Lin
Yee-Shin
YS
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D016627:Oxidopamine / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000187:drug effects / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D013950:Thymus Gland / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology
D000975:Antioxidants; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D016627:Oxidopamine; D013482:Superoxide Dismutase; D020169:Caspases