THE ROLE OF P27(KIP-1) IN THE IN-VITRO DIFFERENTIATION OF MURINE KERATINOCYTES

We have studied the regulation of cyclins and cyclin-dependent kinase activities during differentiation of primary mouse keratinocytes. Diff erentiation was induced by placing primary murine keratinocytes into s uspension culture, under conditions which prevent cells from attaching to any surface. This treatment induces synthesis of keratin 1, one of the earliest known markers of keratinocyte differentiation, and also results in a profound change in the regulation of G(1) and S-phase cyc lins and their associated proteins as well as their activities, The pl acement of cells in suspension culture reduced cyclin A, D1, and E kin ase activity within 6 h, accompanied by the cessation of DNA synthesis , K1 mRNA levels were observed to increase after this period, supporti ng the hypothesis that cell cycle withdrawal precedes the differentiat ion program, Our data further revealed that the p27(kip1) protein leve l and associated cyclin-dependent kinase inhibitory activity increased when keratinocytes were induced to differentiate, Pretreatment of adh erent keratinocytes with p27(kip1) antisense oligonucleotides dramatic ally reduced the accumulation of p27(kip1) protein upon subsequent sus pension culturing and prevented the onset of differentiation independe ntly of the loss of cyclin-dependent kinase activities, Although antis ense oligonucleotide treatment inhibited differentiation, it did not p revent growth arrest, Therefore, the differentiation of primary mouse keratinocytes required a function of Kip other than the inhibition of cyclin-associated activities, and we suggest that this requirement may reflect a novel Rb kinase activity present in Kip immune complexes, w hich is dependent on the presence of cyclin D3. Thus, the placement of keratinocytes in suspension induces a program that includes loss of c yclin activity, which is linked to terminal growth arrest, and an indu ction of p27(kip1), which is linked to the differentiation program.