p27Kip1 and p18INK4cin T-47D cell cycle arrest
- Mike Davies1
© Current Science Ltd 2000
Published: 1 December 2000
KeywordsCDK cell cycle cyclin p18INK4c p27Kip1 progestin T-47D
Progesterone plays an important role in the regulation of proliferation and differentiation in the normal breast by cell-cycle phase-specific actions. Progestin treatment of breast cancer cells in vitro results in transient stimulation followed by inhibition of proliferation. These effects are specific to the G1 phase of the cell cycle, implicating G1 cyclins, CDKs and CDK inhibitors as mediators. Cell cycle arrest is preceded by decreased cyclin abundance and CDK activity and increased association of p27Kip1 with Cdk4-containing complexes and cyclin E-Cdk2 complexes.
To investigate the contribution of G1 cell cycle proteins to progestin-mediated inhibition of human breast cancer cell proliferation, including the relative importance of p21Cip1, p27Kip1 and members of the INK4 family of CDK inhibitors.
This paper clearly illustrates the complex nature of G1 cell cycle regulation and demonstrates the need to consider not just relative levels of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors, but also their different associations and activities. The interplay between the Kip/Cip and INK4 families of CDK inhibitors is particularly interesting. Whether the control mechanisms explored here are typical of normal cells in vivo, or are modulated by the use of cancer cells in tissue culture, is yet to be discerned.
T-47D cells were grown in culture and treated with synthetic progestin ORG2058 or vehicle control for 24 h prior to preparation of cell lysates. Immunoprecipitation, gel filtration chromatography, western blotting and 2D gel electrophoresis were used in combination with recombinant proteins, kinase assays and RNase protection assays to assess the relative levels of cell cycle related mRNA, protein, protein-protein interaction and CDK activity.
Following ORG2058 treatment, cyclin E-Cdk2 is complexed with p27Kip1 in a ~200 kDa, kinase-inactive form. The majority of cyclin D1/-Cdk4/6 complexes are bound by p27Kip1, and to a lesser extent p21Cip1, without progestin treatment, with some increase in these complexes following treatment. The inability of p27Kip1 to inhibit Cdk4 was confirmed in vitroby incubation with recombinant hexahistidine-tagged recombinant p27.
Analysis of p27Kip1 isoforms in treated and untreated cells indicated that modification of p27Kip1 does not account for the effect of ORG2058 in cyclin-CDK complexes. However, addition of ~34-182 pM recombinant His6-p27 to cell lysates in vitro was capable of mimicking progestin-stimulated inhibition of cyclin E-Cdk2. Recombinant His6-p27 was also capable of recapitulating the shift in size from ~120 kDa to ~200 kDa seen following ORG2058 treatment.
Whilst progestin treatment results in decreased levels of cyclins D1 and D3, neither this nor the increased association with p27Kip1 is sufficient to explain the ablation of Cdk4 kinase activity. Levels of p18INK4c and p15INK4b increased in cells following ORG2058 treatment. Treatment also results in association of Cdk4 and Cdk6 with p18INK4c. Cdk6 was found to be bound to p18INK4c following progestin treatment, but a significant amount of Cdk6 was bound to p18INK4c even in untreated cells.One mode of action of p18INK4c is to inhibit Cdk2 by releasing p27Kip1 from Cdk4/6 complexes. Progestins were shown to cause a significant, but transient increase in p18INK4c mRNA, suggesting the increase seen in p18INK4c protein was due to transcriptional stimulation. The same was not true for other members of the INK4 family or p27Kip1.
The inhibition of T-47D cell proliferation seen on long-term treatment with progestins is the result of a series of changes in protein expression and interaction among the G1 cyclins, CDKs and CDK inhibitors. Whilst cyclin E-Cdk2 complexes are inhibited by association with p27Kip1, the same is not true for cyclin D-Cdk4/6 complexes. Rather, in addition to decreases in cyclin D levels, Cdk4/6 activity is inhibited by association with p18INK4c which displaces p27Kip1. The subsequent redistribution of p27Kip1contributes to inhibition of Cdk2.