Synchronization of Cultured Vascular Smooth Muscle Cells Following Reversal of Quiescence Induced by Treatment with the AntioxidantN-Acetylcysteine

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 antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/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/G₁DNA 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-mybgene expression during mid and late G₁phase, 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 G₀quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.     

A Passiflora homolog of a D-type cyclin gene is differentially expressed in response to sucrose, auxin, and cytokinin

In higher plants, one of the major components of developmental processes is cell division. The cell division cycle in plants is controlled by cyclins and cyclin-dependend kinases. Nutrient and hormonal signals can influence the roles that D-type cyclins play in the G1-to-S phase transition. Auxins and cytokinins are long known to be important plant hormones controlling plant growth. Additionally, as sucrose is the major transported carbon source in higher plants, it is possible that it plays a major role in cell division. To access the molecular aspects of the effect of auxin, cytokinin and sucrose on the regulation of cell cycle machinery and plant development, we cloned a Passiflora morifolia putative homolog to a D-type cyclin, PmCYCD1, which showed high sequence similarity to other known plant D-type cyclins. We examined the expression patterns of PmCYCD1 during callus induction and growth in in vitro conditions. We observed incremented expression levels of PmCYCD1 correlated to increasing concentrations of sucrose, α-naphthalene acetic acid and 6-benzyladenine in the culture medium. Additionally, the results of in situ hybridization experiments indicated a dynamic spatial expression pattern for PmCYCD1 during callus growth.