Distinct roles for p53 transactivation and repression in preventing UCN-01-mediated abrogation of DNA damage-induced arrest at S and G2 cell cycle checkpoints.

The topoisomerase I inhibitor SN38 arrests cell cycle progression primarily in S or G(2) phases of the cell cycle in a p53-independent manner. The Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), overcomes both S and G(2) arrest preferentially in cells mutated for p53, driving cells through a lethal mitosis and thereby enhancing cytotoxicity. The mechanism by which p53 maintains S and G(2) arrest was investigated here. The p53 wild-type MCF10A cells were arrested in S phase by incubation with SN38 for 24 h. Subsequent incubation with UCN-01 failed to abrogate arrest. To examine the impact of p53, MCF10A cells were developed, which express the tetramerization domain of p53 to inhibit endogenous p53 function. These cells were attenuated in SN38-mediated induction of p21(WAF1), and UCN-01 induced S, but not G(2) progression. In contrast, MCF10A cells expressing short hairpin RNA to ablate p53 expression underwent both S and G(2) phase progression with UCN-01. The difference in G(2) progression was attributed to p53-mediated gene repression; the MCF10A cells expressing the tetramerization domain retained p53 protein and repressed both cyclin B and Chk1, while cells ablated for p53 did not repress these proteins. Hence, inhibition of p53 activator function permits S phase abrogation, while additional inhibition of p53 repressor function is required for abrogation of G(2) arrest. These studies provide a mechanistic explanation for how this therapeutic strategy can selectively target tumor cells.

Positive and negative regulation of deltaN-p63 promoter activity by p53 and deltaN-p63-alpha contributes to differential regulation of p53 target genes.

Mammary epithelial regeneration implies the existence of cellular progenitors with retained replicative capacity, prolonged lifespan and developmental potency. Evidence exists that deltaN-p63 isoforms preserve these features by modulating p53 activity in basal epithelia. deltaN-p63 mRNA levels decline at the onset of differentiation suggesting that its transcriptional regulation may contribute to the initiation of differentiation. To study transcriptional regulation of deltaN-p63, a 10.3 kbp fragment containing the deltaN-p63 promoter was isolated. We report here that deltaN-p63 is a positive and negative transcriptional target of p53 and deltaN-p63-alpha, respectively. Disruption of p53 activity or expression abolishes the expression of deltaN-p63-alpha. This regulation is mediated by a p53-binding element sufficient to confer these activities to a heterologous promoter. Chromatin immune-precipitation indicates that, in asynchronously growing cells, p53 occupies this element. In response to DNA damage, deltaN-p63-alpha is recruited to this element as transcription of deltaN-p63 declines. Disruption of deltaN-p63-alpha expression had differential effects on the transcriptional regulation of several p53-target genes. These findings indicate that p53 contributes to the preservation of basal epithelia by driving the expression of deltaN-p63 isoforms. These studies also suggest that in response to genotoxic stress, deltaN-p63-alpha mediates the silencing of its own promoter thereby altering the pattern of p53-target gene expression.