BRIEF EXPOSURES OF RESTING FIBROBLASTS TO OKADAIC ACID STIMULATE DNA-SYNTHESIS

To study further the factors providing for cellular quiescence, we use d okadaic acid (OA) at concentrations (0.1, 1, 10 or 100 nM) inhibitin g type 1 and/or type 2A protein phosphatases in mammalian cell culture s. Brief (2 h) exposure of resting (0.2% serum for 72 h) NIH 3T3 mouse fibroblasts to OA with subsequent incubation of cells in a medium wit h 0.2% serum, stimulated DNA synthesis at all concentrations studied. Maximal stimulation was observed following pre-incubation of resting c ells with 10 nM OA. Treatment of cycling cells (10% serum) with OA (2 h pulses at 12 h intervals for 72 h) prevented their exit to the resti ng state on transfer to a medium with 0.2% serum. Brief exposures of r esting cells to OA did not affect the rate of protein synthesis. OA pu lses in the late pre-replicative period had no effect on the entry of serum-stimulated cells into the S phase. Cell fusion experiments with resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 cells, using radioautography with a double-labelling technique, reveal ed that pre-incubation of resting cells with OA for 2 h before and aft er fusion abrogates their ability to suppress the onset of DNA synthes is in the nuclei of proliferating cells in heterodikaryons. The result s indicate that protein phosphatases of type 1 and/or 2A may be involv ed in the growth-arrest machinery that provides for cellular quiescenc e.