N-linked glycoprotein synthesis and transport during G1 are necessary for astrocytic proliferation

The proliferation of astrocytes, purified by a selective detachment technique from mixed glial primary cultures derived from newborn rat cerebrum, was studied. The cells were synchronized by first inducing a quiescent state by removing fetal calf serum (FCS) from the culture medium for 2 days; reversal of the quiescent state by return of serum to the culture medium caused a marked increase in DNA synthesis 12-24 hr later. 2-Deoxyglucose, an inhibitor of dolichol-linked oligosaccharide and thereby N-linked glycoprotein biosynthesis, prevented not only an increase in glycoprotein biosynthesis in G1 phase of the cell cycle but also the burst of DNA synthesis that followed during S phase. Addition of mannose to the culture medium prevented the inhibitions by deoxyglucose of both glycoprotein and DNA syntheses. These data indicated an obligatory relationship in astrocytes between dolichol-linked glycoprotein synthesis and DNA synthesis. To determine whether transport of the newly synthesized glycoproteins to the plasma membrane for incorporation therein or for secretion were necessary for DNA synthesis and astrocytic proliferation, we studied cells treated with monensin, an ionophore for monovalent cations, and an inhibitor of intracellular transport of glycoproteins. The presence of monensin in the first 12 hr after repletion of serum to synchronized astrocytes prevented progression to the S phase and cell proliferation; addition of monensin after the first 12 hr, at the onset of the S phase, had no effect on progression through S phase. Lectin-staining methods combined with fluorescence microscopy demonstrated in monensin-treated cells failure of intracellular glycoproteins to be transported to the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)