Overexpression of acetylcholinesterase inhibited cell proliferation and promoted apoptosis in NRK cells.
OBJECTIVE
To study the potential function of acetylcholinesterase (AChE) in apoptosis through overexpression of AChE in Normal Rat Kidney (NRK) cells.


METHODS
AChE activity was detected by the method of Karnovsky and Roots. Activated caspase-3 was analyzed by Western blotting and immunofluorescence with antibody special to activated caspase-3 fragment. The expression plasmids were constructed in pcDNA3.1 containing AChE gene or a fragment of AChE antisense that were got from RT-PCR. Stable expression cell lines were selected by G418 in cells transfected by lipofection. AChE expression was analyzed by RT-PCR and Western blotting. The proliferation rates of transfected cells were examined by the growth curve and cloning efficiency. MTT assay was used to analyze the cell viability.


RESULTS
The proliferation rate of the cells transfected with AChE was retarded and the cloning efficiency was lower (28.2 %+/-3.1 % and 48.7 %+/-2.1 %) than cells transfected with vector (56.1 %+/-0.3 %) or AChE-antisense (77.7 %+/-2.2 %). After 2 d the various clone types were deprived of serum, the residue cell viability were 10.4 %+/-4.6 % and 12.6 %+/-6.7 % in the cells transfected with AChE, and 27.4 %+/-3.5 % in cells with vector, and 50.3 %+/-7.8 % in cells with AChE-antisense.


CONCLUSIONS
During apoptosis, increase of AChE protein is to inhibit cell proliferation, and then to promote apoptosis in NRK cells.
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology and Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China.
Qi-Huang
QH
Jin
Heng-Yi
HY
He
Yu-Fang
YF
Shi
He
H
Lu
Xue-Jun
XJ
Zhang
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D002455:Cell Division; D002478:Cells, Cultured; D016373:DNA, Antisense / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007668:Kidney / Q000166:cytology* / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014162:Transfection
D016373:DNA, Antisense; D012333:RNA, Messenger; D000110:Acetylcholinesterase