Strokes: an independent study.
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Acute Disease↗
Biomedical subjects
L R Santiago
Publications and source records attributed to L R Santiago.
AF64A-induced changes in N-myc expression in the LA-N-2 human neuroblastoma cell line are modulated by choline and hemicholinium-3.
Due to AF64A's structural similarity to choline, AF64A can selectively affect cholinergic neurons, which possess a high affinity choline transport system for acetylcholine synthesis. The mechanism by which AF64A selectively produces its cytotoxic effect is unknown. However, based on previous studies that demonstrate that DNA lesions produced by AF64A caused premature termination of N-myc transcription in vitro, it is possible that AF64A may affect the transcription of genes necessary for developmental maintenance in cholinergic cells. Using the LA-N-2 cells as a model to study the effects of AF64A in a purely cholinergic system, we investigated the effects of AF64A on the expression of the N-myc gene and monitored cell growth. AF64A produced a maximal decrease in N-myc mRNA with a return to steady state levels at later time points. Moreover, a decrease in cell numbers in AF64A-treated cells was observed, and these cells did not double in number at their respective doubling time as compared to control. In other studies, a causal relationship between a reduction in N-myc and an inhibition of cell growth and replication has been reported. While these studies do not allow us to conclude that AF64A is specific for N-myc, the data do, nevertheless, suggest that AF64A affects cell growth and/or replication by down-regulating the expression of N-myc which is involved in differentiation and cell growth in neuroblastomas. Presence of choline or hemicholinium-3 prevented the AF64A-induced decrease of N-myc levels by competing with, or inhibiting the choline transport mechanism by which AF64A enters the cell, respectively.
Aziridines↗
AF64A-induced cytotoxicity and changes in choline acetyltransferase activity in the LA-N-2 neuroblastoma cell line are modulated by choline and hemicholinium-3.
The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 microM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10(-5), 10(-4) and 10(-3) M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10(-3) M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A.
Animals↗