Cooperation of p27 Kip1 and p18 INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells

Abstract

ABSTRACT The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G 1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27 Kip1 among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27 Kip1 and few were bound to p21 Cip1 . In vitro, recombinant His 6 -p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His 6 -p27 in vitro or p27 Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18 INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18 INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27 Kip1 and p18 INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.