Connecting p63 to Cellular Proliferation: The Example of the Adenosine Deaminase Target Gene

Abstract

An unresolved issue regards the role of p73 and p63, the two homologs of the p53 oncosuppressor gene, in normal cells and in tumor development. Specific target genes for each protein need to be identified and characterized in order to understand the specific role of each protein in tumor initiation and progression as well as in oncosuppression and development. We tested whether p63 is implicated in transcriptional events related to sustaining cell proliferation by transactivation of antiapoptotic and cell survival target genes such as Adenosine Deaminase (ADA), an important gene involved in cell proliferation. We demonstrate that ADA is a direct target gene of p63 isoforms. In human keratinocytes, the rate of proliferation and the high level of ADA transcript diminished upon elimination of p63 by small interfering RNA. Reporter assays and chromatin immunoprecipitation experiments indicate a physical interaction of p63 with the two putative p53 binding sites we identified in the ADA gene. Moreover, in response to p53 stabilization and ∆Np63 downregulation in normal keratinocytes after U.V. treatment, we found a change in the transcriptional pattern of the p53 family target genes, consistent with the different roles played by p53 and p63 in tumor suppression and cellular proliferation. In fact p53 upregulation determined an increase in p21, which in turn mediated the cell cycle arrest, while the downregulation of ∆Np63 determined a marked decrease in ADA transcript. The experiments reported here support the hypothesis that TAp63 and ∆Np63 might contribute to tumor genesis not exclusively by antagonizing p53, but by conferring a proliferative potential on cancer cells through the transactivation of target genes indispensable for cell division, such as the Adenosine Deaminase gene.

Direct targets of the TRP63 transcription factor revealed by a combination of gene expression profiling and reverse engineering

“…Consistent with this observation, a positive effect on cell proliferation has been previously reported in human keratinocytes (Sbisa et al 2006;Truong et al 2006) and in zebrafish epidermis (Lee and Kimelman 2002).…”

p73 and p63 Sustain Cellular Growth by Transcriptional Activation of Cell Cycle Progression Genes

“…1 and 2A and B), they should differentially regulate the subset of G 1 -S and S phase target genes analyzed. Indeed, we previously showed that ADA and FASN are direct p73 and p63, but not p53, target genes (11,12,14). To test the ability of the different p53 family members to regulate the ADA, FASN, POLD2, CCND3, CDC25C, and CDC2 promoters, we performed transactivation assays.…”

“…MCF-7 cells were lysed and extracted as previously described (11). For immunoblotting, the following primary antibodies were used: p53 DO-1 (1:300; Santa Cruz Biotechnology), p53-pAb1801 (1:100; Santa Cruz Biotechnology), p63 antibody 4A4…”

“…MCF-7 cells were cultured in 150 mm culture dishes and synchronized as previously described. At the given time points, proteins were crosslinked to DNA in living nuclei and chromatin immunoprecipitation assay was performed as described (11). The following antibodies were used to immunoprecipitate the DNA-protein complexes: p53 antibody DO-1 (Santa Cruz Biotechnology), p63 antibody H137 (Santa Cruz Biotechnology), p73 antibodies H-79 and C-20 (Santa Cruz Biotechnology), or unrelated control anti-Flag antibody (Sigma).…”

YAP: At the crossroad between transformation and tumor suppression

“…p73 is a member of the p53 family of transcription factors, and it shares many characteristics with its famous cousin (reviewed in refs. [11][12][13][14][15]. It is able to recognize the same consensus sequences on many promoters of genes induced by wild-type p53, which lead to the accumulation of proteins involved in cell cycle arrest, senescence or apoptosis.…”