Efficient down-regulation of cyclin A-associated activity and expression in suspended primary keratinocytes requires p21(Cip1).
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.
Molecular Oncology Program and Experimental Therapeutics Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology, University of South Florida School of Medicine, Tampa FL 33612, USA.
Paul
P
Hauser
Le
L
Ma
Deepak
D
Agrawal
Eric
E
Haura
W Douglas
WD
Cress
W Jackson
WJ
Pledger
D000818:Animals; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D019925:Cyclin A / Q000235:genetics* / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D050684:E2F Transcription Factors; D017353:Gene Deletion; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D008565:Membrane Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C064554:loricrin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D018844:Cyclin-Dependent Kinases