Keratinocyte growth arrest is associated with activation of a transcriptional repressor element in the human cdk1 promoter

In this study we examined the regulation of cdk1 expression in normal human epidermal keratinocytes (HEKs) and neoplastic keratinocytes. Keratinocytes were growth-arrested by allowing the cells to grow to confluence or by treating them with interferon-gamma (IFNgamma) or 12-O-tetradecanoyl phorbol-13-acetate (TPA). RT-PCR and Western blot analysis demonstrated that cdk1 was profoundly reduced in growth-arrested HEKs when compared with dividing HEKs. In contrast, a squamous carcinoma cell line, SCC25, did not growth-arrest in response to growth inhibitors and did not downregulate cdk1 expression. Transfection of HEKs with a reporter gene driven off a 2.5-kb fragment of the human cdk1 promoter indicated that the downregulation of cdkl upon growth arrest was transcriptional. Deletion mapping of the cdk1 promoter indicated that a repressor region was located between -949 - -722 bp. This repressor region was not operative in the SCC25 cells. Examination of DNA:protein binding complexes by gel-shift analysis indicated that nuclear factors from both proliferative and growth-arrested cells bound to the DNA fragment spanning -949- -722 bp. Further analysis revealed that this binding could be resolved into a constitutive and growth arrest-specific complex that bound in a similar fashion to regions spanning -892 - -831 bp and -831 - -774 bp, respectively. The putative growth arrest-specific complex 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, by competition analysis, to regions -874 - -853 bp and -830 - -800 bp. This is the first report of a transcriptional repressor being operative during keratinocyte growth arrest.