Antisense agrin cDNA transfection blocks neuroblastoma cell-induced acetylcholine receptor aggregation when co-cultured with myotubes

A neuroblastoma x glioma hybrid cell line, NG108-15, was able to induce the aggregation of AChRs when co-cultured with myotubes. NG108-15 cells in culture expressed agrin, producing a protein of similar to 220 kDa and a transcript of similar to 8.0 kb. The mRNA encoding the agrin isoform having no amino acid insertion at either the Y or the Z site, namely agrin(0,0), was the only transcript detected in NG108-15 cells when they were cultured alone or co-cultured with myotubes. NG108-15 cells could be induced to differentiate by chemical treatment, and the chemical-induced differentiation of NG108-15 cells increased the level of agrin mRNA expression approximately fourfold while the expression of a housekeeping gene remained relatively unchanged. The increase in agrin expression of differentiated NG108-15 cells paralleled the increase in AChR-aggregating activity of differentiated NG108-15 cells, indicating that the agrin derived from NG108-15 cells could be the receptor-aggregating factor. In addition, we created a stable clonal NG108-15 cell line that was transfected with antisense agrin cDNA and its expression of agrin was abolished, while its AChR-aggregating activity was completely lost when co-cultured with myotubes. This is the first direct demonstration that NG108-15 cell-induced AChR aggregation on cultured myotubes is mediated by neuron-derived agrin.