Abrogation of p27 by cDNA Antisense Suppresses Quiescence (G0 State) in Fibroblasts

Abstract

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. p27Kip1 (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 mitogensensitive 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 serumstimulated 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.

Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells

“…These investigators did not, however, examine possible e ects of overexpression of p27 on di erentiation. Increased expression of p27 can enhance exit from the cell cycle (Hengst and Reed, 1996;Rivard et al, 1996). The ability of p27 to enhance the differentiation of HT29 cells observed in the present studies is consistent with previous evidence that the levels of this protein increase in HL60 promyelocyctic leukemia cells (Hengst and Reed, 1996) or neuroblastoma cells (Kranenburg et al, 1995) that are induced to di erentiate by vitamin D3 or DMSO, respectively.…”

A model for restriction point control of the mammalian cell cycle

“…As a consequence, Kip1 À cells lack the G1pm phase. In simulations (not shown), cells deprived of GF or treated with CHX keep on proliferating, which agrees with the observation that Kip1 À cells are less serum-dependent than Kip1 + cells (Coats et al, 1996;Rivard et al, 1996).…”

p27Kip1-deficient mice exhibit accelerated growth hormone-releasing hormone (GHRH)-induced somatotrope proliferation and adenoma formation

“…It is well documented that p27 restricts entry of cells into S phase by binding and inhibiting the activity of cyclin/ CDK complexes Sherr and Roberts, 1999). Overexpression of p27 blocks cells in G1 while suppression of p27 mRNA translation by antisense oligonucleotides maintains cells in a proliferative state (Coats et al, 1996;Rivard et al, 1996). Although p27 protein levels clearly¯uctuate during progression through the cell cycle, p27 mRNA levels remain relatively constant (Hengst and Reed, 1996).…”