The role ofp53 in retinoic acid-induced differentiation of human germ cell tumors

The retinoids exert potent growth and differentiation effects on normal, embryonic and neoplastic cells. Although retinoids are known to regulate gene transcription through activation of retinoid receptors, the direct target genes of retinoid receptors remain largely unknown. A human embryonal carcinoma (EC) model was employed to determine the importance of p53 in retinoid-mediated differentiation and signaling. The hypothesis was that one mechanism by which retinoids regulate gene expression and mediate beneficial anti-tumor effects is by activation of p53 transactivation activity. RA was found to enhance the transactivation function of p53 in the human EC line NT2/D1. A derived RA-resistant line, NT2/D1-R1, was deficient in this activity, and also exhibited cross-resistance to cisplatin. This suggested a model in which RA and cisplatin signaling pathways impinge upon p53 in human EC cells. Further evidence generated to support this model is the following: (1) RA-induced p53 activity could be restored in NT2/D1-R1 cells by engineered expression of various RARs; (2) inhibition of p53 via dominant/negative suppression or small inhibitory RNA inhibited RA-induced p53 transactivation and blocked the RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid in NT2/D1 cells; (3) inhibition of p53 activity also enhanced survival of NT2/D1 cells following cisplatin treatment. An effort to identify a region of p53 responsible for RA-mediated activation led to the discovery of a 27-residue peptide of p53 (residues 90–116) that conferred basal repression to p53 in NT2/D1 cells. This previously uncharacterized region had the ability to act as a heterologous repressor of transcription factor activity. This region has been named the p53 N-terminal Repression Domain (p53-NRD). Repression mediated by p53-NRD could be relieved by co-treatment with RA and the histone deacetylase inhibitor trichostatin-A (TSA) in NT2/D1, but not NT2/D1-R1 cells. Together, these data indicate that RA activates the intrinsic activation function of p53 by a potentially novel mechanism independent of effects on p53 stability or DNA binding. This activation may be a general mechanism that contributes to RA-mediated G1 arrest and differentiation. In addition, the p53 latency generally associated with human EC is mediated, at least in part, by residues 90–116 of the p53 protein.