Cholinergic differentiation triggered by blocking cell proliferation and treatment with all-trans-retinoic acid

This study determined whether the effect of all-trans-retinoic acid (t-RA) on markers of cholinergic differentiation in a murine septal cell line. SN5 6.B5.G4, differed depending upon the cell's proliferative status. To develo p a model of non-proliferating cells, aphidicolin, a DNA ol-polymerase inhi bitor, was used. Cessation of proliferation by aphidicolin increased intrac ellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transp orter (VAChT) mRNA. Importantly, the response to t-RA differed depending up on proliferative status. Consistent with previous reports, t-RA increased C hAT and VAChT mRNA, ChAT activity and intracellular ACh levels in prolifera ting SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive e ffect of combined treatment was observed on ACh levels: nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs , but not ChAT activity. indeed, aphidicolin treatment completely suppresse d the t-RA-induced increase in ChAT activity observed in proliferating cell s. To explore the response to t-RA in post-mitotic cells, a sequential trea tment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequent ial treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell's proliferative status. (C) 2000 Elsevier Science B.V. All r ights reserved.