Acetylcholinesterase In Apoptosis And G418 Induced Apoptosis Molecular Mechanism

Acetylcholinesterase (AChE) is expressed in a number of cell lines uponinduction of apoptosis by various stimuli, and inhibiting expression of AChE byAChE antisense prevents part of the cells from apoptosis. However, the function ofAChE in apoptosis is still elusive. We made AChE overexpresse in Normal RatKidney (NRK) cells to investigate its possible function. AChE activity was notpresented in living NRK cells, but the AChE protein existed. Upon induction ofapoptosis, apoptotic NRK cells presented AChE activity, and AChE mRNA andprotein also increased. Overexpression of AChE in NRK cells did not induceapoptosis. However, the proliferation of cell lines expressing AChE was retarded, andthe effect was reversed in cell lines overexpressing AChE antisene, in which AChEexpression decreased. Under induction by serum deprivation, the residual cell activityof cells overexpressing AChE was lower than control, and the effect was reversed incell lines expressing AChE antisense. It suggested that AChE had two functions in theprocess of apoptosis: increased AChE protein first inhibited cell proliferation, andthen promoted apoptosis. To detect the possible function of inactive AChE duringapoptosis, NRK cells were induced apoptosis by G418, which is an inhibitor ofprotein synthesis. Apoptotic NRK cells induced by G418 exhibited strong AChEactivity, which was independent of new protein synthesis and capases activity. Inliving NRK cells, AChE protein located in ER, and in apoptotic cells, AChEdistributed over the whole cell. The products of AChE enzymatic reaction observedunder electron microscope were located in nuclei, cytosole, and on the membrane ofsome vacuoles. Expression of AChE without ER location signal (ERLS) did notinduce apoptosis. However, the percentage of apoptotic cells in NRK cells transfectedwith AChE without ERLS was higher than that in NRK cells transfected with intactAChE. Interestingly, cells expressed C-terminal of AChE had the same effect withcells expressed AChE without ERLS, and moreover, AChE directly expressed incytosole had no activity. Base on these experiments, we brought forward a hypothesison AChE in apoptosis: in the process of apoptosis, inactive AChE, which stably stoodin ER in NRK cells, was translocated into cytosole and nuclei, where the C-terminalof AChE interacted with some apoptotic factors and promoted apoptosis, and at thesame time, inactive AChE was transformed into active AChE. To further analyse theinactive AChE protein, we prepared antibody affinity chromatography to isolate thetotal AChE. This method with tacrine affinity chromatography which was to isolateactive AChE provided precondition for analysis of the difference between inactive andactive AChE at the protein level.