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Cell Cycle, Cell Death, and Senescence

Efficient Down-Regulation of Cyclin A-Associated Activity and Expression in Suspended Primary Keratinocytes Requires p21^Cip11

¹ Molecular Oncology Program and
² Experimental Therapeutics Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology, University of South Florida School of Medicine, Tampa FL

Requests for reprints: W.J. Pledger, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa FL 33612. Phone: (813) 979-3887; Fax: (813) 979-3393. E-mail: pledgerw{at}moffitt.usf.edu

When suspended in methylcellulose, primary mouse keratinocytes cease proliferation and differentiate. Suspension also reduces the activity of the cyclin-dependent kinase cdk2, an important cell cycle regulatory enzyme. To determine how suspension modulates these events, we examined its effects on wild-type keratinocytes and keratinocytes nullizygous for the cdk2 inhibitor p21^Cip1. After suspension of cycling cells, amounts of cyclin A (a cdk2 partner), cyclin A mRNA, and cyclin A-associated activity decreased much more rapidly in the presence than in the absence of p21^Cip1. Neither suspension nor p21^Cip1 status affected the stability of cyclin A mRNA. Loss of p21^Cip1 reduced the capacity of suspended cells to growth arrest, differentiate, and accumulate p27^Kip1 (a second cdk2 inhibitor) and affected the composition of E2F DNA binding complexes. Cyclin A-cdk2 complexes in suspended p21^+/+ cells contained p21^Cip1 or p27^Kip1, whereas most of the cyclin A-cdk2 complexes in p21^−/− cells lacked p27^Kip1. Ectopic expression of p21^Cip1 allowed p21^−/− keratinocytes to efficiently down-regulate cyclin A and differentiate when placed in suspension. These findings show that p21^Cip1 mediates the effects of suspension on numerous processes in primary keratinocytes including cdk2 activity, cyclin A expression, cell cycle progression, and differentiation.

Key Words: keratinocytes • p21^Cip1 • cyclin A • suspension • differentiation