KERATINOCYTE GROWTH ARREST IS ASSOCIATED WITH ACTIVATION OF A TRANSCRIPTIONAL REPRESSOR ELEMENT IN THE HUMAN CDK1 PROMOTER

In this study Lye examined the regulation of cdk1 expression in normal human epidermal keratinocytes (HEKs) and neoplastic keratinocytes. Ke ratinocytes were growth-arrested by allowing the cells to grow to conf luence or by treating them with interferon-gamma (IFN gamma) or 12-O-t etradecanoyl phorbol-13-acetate (TPA). RT-PCR and Western blot analysi s demonstrated that cdk1 was profoundly reduced in growth-arrested HEK s when compared with dividing HEKs. In contrast, a squamous carcinoma cell line, SCC25, did not growth-arrest in response to growth inhibito rs and did not downregulate cdk1 expression. Transfection of HEKs with a reporter gene driven off a 2.5-kb fragment of the human cdk1 promot er indicated that the downregulation of cdk1 upon growth arrest was tr anscriptional. Deletion mapping of the cdk1 promoter indicated that a repressor region was located between -949--722 bp. This repressor regi on was not operative in the SCC25 cells. Examination of DNA:protein bi nding complexes by gel-shift analysis indicated that nuclear factors f rom both proliferative and growth-arrested cells bound to the DNA frag ment spanning -949--722 bp. Further analysis revealed that this bindin g could be resolved into a constitutive and growth arrest-specific com plex that bound in a similar fashion to regions spanning -892--831 bp and -831--774 bp, respectively. The putative growth arrest-specific co mplex was not found in contact-inhibited fibroblasts and was found at very low levels in SCC25 cells, indicating that the putative repressor binding was growth arrest-specific and possibly keratinocyte-specific . The binding complexes bound to these two fragments were localized, b y competition analysis, to regions -874--853 bp and -830--800 bp. This is the first report of a transcriptional repressor being operative du ring keratinocyte growth arrest. (C) 1998 Wiley-Liss, Inc.