UP-REGULATION OF P27(KIP1), P21(WAF1 CIP1) AND P16(INK4A) IS ASSOCIATED WITH, BUT NOT SUFFICIENT FOR, INDUCTION OF SQUAMOUS DIFFERENTIATION/

Irreversible growth arrest is an early and integral part of squamous c ell differentiation in normal human epidermal keratinocytes (NHEKs) an d is assumed to be linked to the control of expression of differentiat ion-specific genes. In this study, we examine the link between the mol ecular events associated with growth arrest and the expression of diff erentiation genes. NHEKs that have been induced to undergo growth arre st and differentiation by suspension culture contain populations in bo th G(1) and G(2)/M of the cell cycle. The irreversible growth arrest s tate in NHEKs is characterized by an accumulation of the hypophosphory lated forms of Rb and p130, with subsequent down-regulation of levels of Rb, up-regulation of p130 and associated down-regulation of E2F-reg ulated genes such as cyclin A. These events correlate with an inhibiti on of G(1) cdk activity, mediated in part by an increase in the cdk in hibitors p21(WAF1/Cip1), p27(Kip1) and p16(Ink4a). Flow cytometric and immunoblot analysis demonstrated that the timing of the up-regulation of p27, p16 and p130 corresponds closely with the induction of the sq uamous-specific genes cornifin Phi (SPRR-1) and transglutaminase type I, suggesting a close link between control of growth arrest and differ entiation. However, growth arrest induced by over-expression of p27, p 21 or p16 by recombinant adenovirus is not sufficient to induce expres sion of the differentiation genes, or to invoke the pattern of cell cy cle regulatory protein expression characteristic of the differentiatio n-specific irreversible growth arrest. We conclude that growth arrest mediated by activation of the Rb pathway is not sufficient to trigger terminal squamous differentiation and additional signals which can be generated during suspension culture are required to promote the comple te differentiation program.