When animal cells are exposed to stressful conditions, the tumor suppressor protein p53 restrains growth by promoting an arrested cell cycle or initiating a cell death program. How these distinct fates are specified through the action of a single protein is not known. To study its functions in vivo we produced a targeted mutation at the Drosophila p53 (Dmp53) locus. We show that Dmp53 is required for damage-induced apoptosis but not for cell-cycle arrest. Dmp53 function is also required for damage-induced transcription of two tightly linked cell death activators, reaper and sickle. When challenged by ionizing radiation, Dmp53 mutants exhibit radiosensitivity and genomic instability. Hence, elevated mutant loads were not caused by defective checkpoint functions but instead correlated with failures in p53-associated cell death. Our studies support the notion that core ancestral functions of the p53 gene family are intimately coupled to cell death as an adaptive response to maintain genomic stability.T he p53 tumor suppressor limits oncogenesis through activities that govern adaptive responses to stress. As such, p53 is thought to function as a ''guardian of the genome'' that becomes mutated or altered in most human cancers (1, 2). When cells are stressed by exposure to genotoxic agents, radiation, hypoxia, or inappropriate oncogene activation (1-3), p53 restrains growth through activities that reversibly arrest the cell cycle or promote apoptosis (4, 5), but how these alternative fates become specified through the action of this tumor suppressor is not understood. The primary mechanism of p53 action clearly involves transcriptional regulation (6-8), although other activities might contribute to accessory functions (9). The protein requires at least three functional domains to regulate downstream target genes but many mutations commonly found in human cancers map to the DNA-binding domain (10-13). A major effort in cancer biology is focused toward understanding downstream effectors of p53 function. The p53 target gene, p21, acts directly as a cyclindependent kinase inhibitor (14-16) and appears largely responsible for p53-dependent G 1 ͞S arrest. The in vivo downstream targets responsible for p53-mediated apoptosis, however, are less well defined. Important candidates for in vivo death effectors include members of the bcl2 family, bax (17), noxa (18), puma (19,20), and the death receptor fas (21,22). In many cases, regulated expression of these genes is context-specific and whether there exists a generic p53 apoptosis program for all cells is not known. Genome-wide searches for p53-responsive genes have also been reported, but it is not yet clear whether these loci represent direct or indirect targets (23,24).A Drosophila homolog of p53, Dmp53, was recently identified (25-27). Like mammalian counterparts (12, 13, 28-31), Dmp53 has a well conserved DNA-binding domain with a transcriptional activation domain at its N terminus, and an oligomerization domain at its C terminus. In previous studies, forced expression of ...

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Retinoic acid (RA), a derivative of vitamin A, critically controls brain patterning and neurogenesis during embryogenesis, and is known to regulate morphological differentiation of catecholaminergic neuronal cells. In this study, we investigated whether the retinoic acid receptor (RAR), a transcription factor specifically activated by all-trans-RA, could directly regulate transcription of tyrosine hydroxylase (TH), the first and ratelimiting step in the catecholamine biosynthesis pathway. First, treating TH-expressing human neuroblastoma SK-N-BE(2)C cells with all-trans RA resulted in an approximately 1.7-fold increase in endogenous TH mRNA expression, as determined by real-time PCR analysis. Second, when SK-N-BE(2)C cells were transiently co-transfected with the TH promoter-luciferase reporter construct, reporter gene expression was prominently activated by RAR in a ligand-dependent manner. Third, we identified a putative RAR responsive cis-regulatory element at ) 1500 to ) 1487 bp in the TH upstream promoter region by deletional and site-directed mutational analysis. Finally, we demonstrated that this putative motif directly interacts with RAR protein in a sequence-specific manner by means of an electrophoretic mobility shift assay. Taken together, our results indicate that the TH gene may be a direct downstream target of the RA signaling pathway and that RAR is able to activate TH transcription through interaction with an upstream sequence motif residing at ) 1500 to ) 1487 bp.

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