Critical role for p27^Kip1 in cell cycle arrest after androgen depletion in mouse mammary carcinoma cells (SC-3)

Abstract

The molecular mechanisms underlying androgen-regulated cancer growth and the frequent development of refractoriness to endocrine therapy remain unknown. In this study functional and quantitative alterations in cell cycle regulators after androgen depletion were examined in androgen-dependent mouse mammary carcinoma cells (SC-3) as a model system to clarify the initial response of cancer cells to anti-androgen therapy. FACS analysis of SC-3 cells cultured with or without 10⁻⁷ M testosterone revealed that suppression of cell growth after hormone withdrawal was due to G1 arrest. Although cyclin D1/Cdk4 activity decreased along with a reduced level of cyclin D1 protein, this was a later event (48–72 h) than the G1 arrest (24 h). Taken together with the results that constitutive expression of cyclin D1 in SC-3 cells did not overcome the growth suppression following androgen depletion, the existence of an alternative pathway(s) causing G1 arrest was suggested. Cyclin E/Cdk2 and cyclin A/Cdk2 activities decreased significantly at 24 h without apparent changes in the amounts of Cdk2, cyclin E or cyclin A. Among various Cdk inhibitors (CKIs) examined, p27^Kip1 was upregulated at both mRNA and protein levels at 24 h after androgen depletion. In addition, immunoprecipitation-Western analysis showed that the amount of p27^Kip1 associated with Cdk2 complexes increased as early as 24 h. These results suggest that p27^Kip1 CKI is a critical target in the initial response of cancer cells to androgen depletion and plays a key role in Cdk2 inactivation through association with the kinase complex, leading to cell cycle arrest.