Synchronization of cultured vascular smooth muscle cells following reversal of quiescence induced by treatment with the antioxidant N-acetylcysteine

Abstract

Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidant N-acetylcysteine (NAC) inhibits constitutive NF-kappa B/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [3H]thymidine and G0/G1 DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-myb gene expression during mid and late G1 phase, respectively, and induction of histone gene expression and [3H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb, and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G0 quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.