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

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 G₁ cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27^Kip1among 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 His₆-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 His₆-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.

ACKNOWLEDGMENTS

We thank David Parry and Emma Lees for the INK4 antibodies, Owen Prall for helpful discussion and critical evaluation of the manuscript, and the following colleagues for reagents and advice: Boris Sarcevic, Amanda Mawson, Bev Warner, Grant Macarthur, Roger Daly, and Steve Coats.

This work was supported by grants from the New South Wales Cancer Council and the National Health and Medical Research Council of Australia. A.S. is the recipient of an Australian Postgraduate Award.