Quiescent astroglia in long-term primary cultures re-enter the cell cycle and require a non-sterol isoprenoid in late G1

Abstract

Astroglia proliferate during brain growth, and can divide again later, particularly during astrogliosis. We investigated whether astroglia in primary cultures of newborn rat brain similarly achieve a state of prolonged quiescence which enables re-entry into the cell division cycle. In cultures after 2 months, cell number plateaued and there were sharp decreases in [3H]thymidine incorporation (70 +/- 5 vs 4 +/- 0.5 cpm/micrograms protein/h at 30 and 60 days, respectively) and in percentages of cell nuclei incorporating bromodeoxyuridine (BrDU) (from 46 +/- 6% to less than 1%). Replating at 10(4) cells/cm2 yielded secondary cultures which synthesized DNA actively. Forty-eight hours of serum deprivation at 2-3 days from subculturing, followed by addition of 10% serum (time 0), resulted in a return to quiescence which persisted until 12 h (G0 + G1). By 20 h (S phase), there were abrupt increases in DNA synthesis (5-fold) and in BrDU-labeled nuclei (from 19 +/- 2 to 76 +/- 8%) and the percentage of glial fibrillary acidic protein (GFAP)-positive cells declined to 14 +/- 2%. Three days later, GFAP-positive cells numbered around 80%. Cell cycling after prolonged quiescence, in a manner similar to that in early astroglial cultures, required a non-sterol derivative of mevalonate in late G1. These data confirm that astroglia in primary cultures, like their counterparts in vivo, have a flexible capacity to enter and depart from quiescence, and most importantly, provide a system for examining regulation of this process.