Effects of acetylcholinesterase gene silencing on its activity in cultured human skeletal muscle.
In spite of several reports demonstrating that acetylcholinesterase (AChE [EC 3.1.1.7]) expression is importantly regulated at the level of its mRNA, we still know little about the relationship between AChE mRNA level and the level of mature, catalytically active enzyme in the cell. Better insight into this relationship is, however, essential for our understanding of the molecular pathways underlying AChE synthesis in living cells. We have approached this problem previously (Grubic et al., 1995; Brank et al., 1998; Mis et al., 2003; Jevsek et al., 2004); however, recently introduced small interfering RNA (siRNA) methodology, which allows blockade of gene expression at the mRNA level, opens new possibilities in approaching the AChE mRNA-AChE activity relationship. With this technique one can eliminate AChE mRNA in the cell, specifically and at selected times, and follow the effects of such treatment at the mature enzyme level. In this study we followed AChE activity in siRNA-treated cultured human myoblasts. Our aim was to find out how the temporal profile of the AChE mRNA decrease is reflected at the level of AChE activity under normal conditions and after inhibition of preexisting AChE by diisopropyl phosphorofluoridate (DFP).AChE activity was determined at selected time intervals after siRNA treatment in both myoblast homogenates and in culture medium to follow the effects of siRNA treatment at the level of intracellular AChE synthesis and at the level of AChE secreted from the cell.
Mis
Katarina
K
Institute of Pathophysiology, University of Ljubljana, Slovenia SI-1000.
Mars
Tomaz
T
Golicnik
Marko
M
Jevsek
Marko
M
Grubic
Zoran
Z
D000110:Acetylcholinesterase / Q000235:genetics*; D002478:Cells, Cultured; D020868:Gene Silencing; D006801:Humans; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger; D000110:Acetylcholinesterase