Evaluation of anti-Fas ligand-induced apoptosis and neural differentiation of PC12 cells treated with nerve growth factor using small interfering RNA method and sampling by microdialysis.
The small interfering RNA (siRNA) method is an effective technique for silencing gene expression and is a useful tool for screening the gene functions in drug discovery. Our study found that nerve growth factor (NGF) can increase the cell viability of PC12 cells and that NGF induction up-regulates the expression of Bcl-2 detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). To further investigate the role of Bcl-2 expression in NGF-treated PC12 cells, the plasmid of Bcl-2 siRNA was then transfected into PC12 cells. Moreover, to investigate and continuously monitor the real-time dynamic neurotransmitter release, and to compare with the time course of Bcl-2 expression, a liquid chromatography coupled with electrochemical detection (LC-ED) and with a microdialysis device was used. After 6h of NGF being added to the PC12 cell culture medium, the dopamine (DA) concentrations were significantly increased (P<0.05). This result is simultaneously compatible with the up-regulated messenger RNA (mRNA) expressions of tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), and Bcl-2 by RT-PCR. Using the Bcl-2 siRNA method, our data revealed that NGF can inhibit Fas ligand (FasL)-induced apoptosis in PC12 cells through the activation of Bcl-2. The in vitro observation further demonstrated that NGF can stimulate the neurite development in PC12 cells through the activation of Bcl-2. Moreover, the DA concentrations of NGF induction were decreased specifically by Bcl-2 siRNA (P<0.05). In sum, our data support that NGF prevents Fas-induced apoptosis, facilitates neural differentiation, promotes dendritic formation, and increases DA release in PC12 cells through activation of Bcl-2.
Chiou
Shih-Hwa
SH
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan.
Kao
Chung-Lan
CL
Chang
Yu-Lih
YL
Ku
Hung-Hai
HH
Tsai
Yung-Jen
YJ
Lin
Han-Tso
HT
Yen
Chih-Ju
CJ
Peng
Chi-Hsien
CH
Chiu
Jen-Hwey
JH
Tsai
Tung-Hu
TH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation; D002470:Cell Survival; D004298:Dopamine / Q000378:metabolism; D053222:Fas Ligand Protein / Q000494:pharmacology*; D017551:Microdialysis; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats
D053222:Fas Ligand Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D020932:Nerve Growth Factor; D053148:Caspase 3; D004298:Dopamine