Cl- channel blockers inhibit transition of quiescent (G0) fibroblasts into the cell cycle

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl- channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl- channel blocker 5-N-2-(3-phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4-aminopyridine (4-AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0-to-G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10-14 h. NPPB applied only at 0-8 or 8-14 h after serum addition inhibited entry into S phase. Cl- permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki-67-negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl- channel blockers (NPPB and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS]) but not the K+ channel blocker (4-AP) significantly decreased the rate of reduction in number of Ki-67-negative cells. These data indicate that an increase in Cl- permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint.