Regulated expression system for GD3 synthase cDNA and induction of differentiation in Neuro2a cells

It was reported recently by our group that the transfectionof GD3 synthase cDNA into Neuro2a cells, a neuroblastoma cellline, caused cell differentiation with neurite sprouting (Kojimaet al, 1994; J. Biol Chem., 269, 30451–30456). To furtherexplore this phenomenon in detail, we applied tet-racycline-regulatedsystem to control the expression of GD3 synthase cDNA in Neuro2acells. Under this system, the process of Neuro2a cell differentiationwas rather slow, about 3 weeks of cell culturing in the absenceof tetracy-cline was required for most cells to extend the neurite-likestructures. The RNase protection assay indicated that the mRNAof GD3 synthase gene was first detected between 4 h and 8 hafter the gene was activated and kept at approximately the samelevel through the process. Furthermore, time-course analysisof total ganglioside expressions has shown that GD3 and GT1bgangliosides appeared on the cell surface early in the processand reached the maximum level around day 6. We also found thatthe amounts of GD3 and GT1b on the cell surface started to decreaseafter day 6 and returned gradually to the basal values after3 weeks. On the other hand, GQ1b and GD1b were started to besynthesized at early stage and the amounts were continuouslyto increase through the whole Neuro2a morphological change process.In addition, time-course analysis by flow cytometry method forGD3 and GQ1b suggested that the conversions of simple gangliosidesto more complex gangliosides may be required to induce the Neuro2adifferentiation. Our results indicated that the combinationof cDNA transfection and regulated gene expression is a powerfultool to study the function of glycolipids and should have ageneral application to the glycobiology field. Neuro2a GD3 synthase gene tetracycline regulated system ganglioside neurite-like structures