Efficient Down-Regulation of Cyclin A-Associated Activity and Expression in Suspended Primary Keratinocytes Requires p21Cip111This work was supported by the Cortner-Couch Endowed Chair for Cancer Research (W. J. P.) and NIH Grants CA73780 (W. J. P.) and CA90489 (W. D. C.)

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 p21Cip1. 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 p21Cip1. Neither suspension nor p21Cip1 status affected the stability of cyclin A mRNA. Loss of p21Cip1 reduced the capacity of suspended cells to growth arrest, differentiate, and accumulate p27Kip1 (a second cdk2 inhibitor) and affected the composition of E2F DNA binding complexes. Cyclin A-cdk2 complexes in suspended p21+/+ cells contained p21Cip1 or p27Kip1, whereas most of the cyclin A-cdk2 complexes in p21−/− cells lacked p27Kip1. Ectopic expression of p21Cip1 allowed p21−/− keratinocytes to efficiently down-regulate cyclin A and differentiate when placed in suspension. These findings show that p21Cip1 mediates the effects of suspension on numerous processes in primary keratinocytes including cdk2 activity, cyclin A expression, cell cycle progression, and differentiation.