p53 is phosphorylated at the carboxyl terminus and promotes the differentiation of human HaCaT keratinocytes

The p53 phosphoprotein acts as a tumor-suppressor gene product through the inhibition of cell growth and induction of apoptosis in a transcription-dep endent manner. These functions require p53 activation through different bio chemical postranslational modifications. Given the relevance of this protei n in ultraviolet light-induced carcinogenesis, whose targets are primarily skin keratinocytes, we studied the functions of p53 in epidermal cell diffe rentiation. We selected HaCaT cells, a human keratinocyte cell line bearing point-mutated, transcriptionally inactive, but highly stable p53, which fa cilitates immunochemical and biochemical analysis. In addition, a reliable in vitro differentiation system has been developed with these cells (Parami o et al. Oncogene 17.949, 1998). We report that during HaCaT differentiatio n there is a loss of immunoreactivity of p53 against antibodies that specif ically recognize epitopes located at the carboxyl terminus of the protein. Because treatment with phosphatase restores this immunoreactivity, we concl ude that p53 is phosphorylated at the carboxyl terminus during keratinocyte differentiation. This biochemical modification has been associated with th e transcriptional activation of the molecule, and because p53 is involved i n differentiation processes in other cell types, we investigated the potent ial functions of p53 during epidermal differentiation. To this end, we gene rated HaCaT clones expressing a murine temperature-sensitive p53 (Mp53ts) b y transfection because the endogenous p53 is not functional even with phosp horylation. We characterized the expression and effects of the transfected protein in different selected clones. The ultraviolet-light response of the se clones was restored, demonstrating the functionality of Mp53ts in these cells. We also observed that, with induction of differentiation, Mp53ts tra nsfected cells differentiate faster than the parental or vector-transfected control cells, demonstrating that p53 promotes epidermal differentiation. The sustained expression of p53 in differentiating cells leads to massive c ell death and detachment, a phenomenon that may be similar to epidermal ter minal differentiation. In addition, we observed that the expression of p53- dependent genes such as p21(waf/cip1) and mdm2 (which are known to particip ate in epidermal differentiation) increases during HaCaT differentiation, i .e., in a p53-independent manner. (C) 2000 Wiley-Liss, Inc.