Abrogation of p27(Kip1) by cDNA antisense suppresses quiescence (G(0) state) in fibroblasts

Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes, p27(Kip1) (p27) is a Cdk inhibitor that blocks, in vitro, the activity of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2 as well as cyclin A-Cdk2, a complex active during S phase, The level of p27 protein expression, usually high in G(0)/G(1) resting cells, declines as cells progress toward S phase and enforced expression of p27 in fibroblasts causes G(1) arrest. This situation prevails in CCL39, a Chinese hamster lung fibroblast cell line (this report), However, in addition to p27, several other Cdk inhibitors known to alter G(1) progression coexist in most mammalian cells, To investigate the specific contribution of p27 in the control of the mitogen-sensitive G(0)/G(1) arrest, we specifically reduced its synthesis by expressing a full-length p27 antisense cDNA in CCL39 cells. Interestingly, reduction of up to 90% of p27 protein expression increased both basal and serum-stimulated gene transcription of cyclin D1, cyclin A, dihydrofolate reductase, and DNA synthesis reinitiation. Moreover, overexpression of this antisense allows cells to grow for several generations in a serum-free medium supplemented with insulin and transferrin only, thus suggesting that p27-depleted cells cannot exit the cell cycle. These effects were fully reversed by coexpression of a plasmid encoding p27 sense. We conclude that p27, by setting the level of growth factor requirement, plays a pivotal role in controlling cell cycle exit, a fundamental step in growth control.