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

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Abstract
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.