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 approximately 220 kDa and a transcript of approximately 8.0 kb. The mRNA encoding the agrin isoform having no amino acid insertion at either the Y or the Z site, namely agrin0.0, 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.
Pun
S
S
Department of Biology, Hong Kong University of Science and Technology, China.
Tsim
K W
KW
D018171:Agrin / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016375:Antisense Elements (Genetics) / Q000235:genetics*; D018920:Coculture Techniques; D018076:DNA, Complementary / Q000235:genetics*; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011940:Receptor Aggregation / Q000502:physiology*; D011950:Receptors, Cholinergic / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018171:Agrin; D016375:Antisense Elements (Genetics); D018076:DNA, Complementary; D011950:Receptors, Cholinergic