Mammary epithelial regeneration implies the existence of cellular progenitors with retained replicative capacity, prolonged lifespan and developmental potency. Evidence exists that DN-p63 isoforms preserve these features by modulating p53 activity in basal epithelia. DN-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 DN-p63, a 10.3 kbp fragment containing the DN-p63 promoter was isolated. We report here that DN-p63 is a positive and negative transcriptional target of p53 and DN-p63-a, respectively. Disruption of p53 activity or expression abolishes the expression of DN-p63-a. 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, DN-p63-a is recruited to this element as transcription of DN-p63 declines. Disruption of DN-p63-a 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 DN-p63 isoforms. These studies also suggest that in response to genotoxic stress, DN-p63-a mediates the silencing of its own promoter thereby altering the pattern of p53-target gene expression.

We assessed the anti-mutagenic and anti-promotion properties of two flavones, apigenin and robinetin, and of indole-3-carbinol, because these compounds have been reported in vegetables, the consumption of which has been associated with reduced rates of cancer. However, the active components of these foods and their effects on carcinogenesis have not been established. Anti-mutagenicity was determined in the Salmonella typhimurium assay by measuring the effects of the test compounds on bacterial mutagenesis induced by methyl-nitrosourea (MNU), methyl-n-nitro-N-nitrosoguanidine (MNNG), benzo[a]pyrene (BaP) or 2-aminoanthracene (2-AA). Inclusion of apigenin resulted in a 62% and a 43% inhibition of mutagenicity with 13 nmol of 2-AA and 30 nmol BaP respectively. Robinetin caused an 87% inhibition of mutagenicity by 2-AA, but indole-3-carbinol had little or no effect on the mutagenicity of any of the compounds. None of the three compounds inhibited mutagenesis by MNU or MNNG and none were mutagenic or toxic when tested in the absence of mutagenic compounds at doses up to 20 micrograms/plate. Anti-promotion properties were assessed by measuring the effects of apigenin, robinetin and indole-3-carbinol on induction of ornithine decarboxylase activity (ODC) in mouse epidermis by 17 nmol 12-O-tetradecanoyl phorbol-13-acetate (TPA). Pretreatment of the skin half an hour before TPA with apigenin, robinetin, butylated hydroxyanisole, 13-cis-retinoic acid (all at 50 mumol) or di-fluoromethylornithine (1.6 mumol) inhibited ODC induction at 6 h after TPA by 67-80%. Pretreatment with 50 mumol indole-3-carbinol caused a 78% elevation in the TPA induction at this time. Dose response measurements were conducted with apigenin, indole-3-carbinol and robinetin. Inhibition by 30-90% of TPA-induced ODC was observed at 6 h after TPA in mice pretreated with 12.5-100 mumol apigenin. Pretreatment with 37.5 or 50 mumol indole-3-carbinol or 0.5, 12.5 or 25 mumol robinetin resulted in elevated induction of epidermal ODC by TPA at 6 h after TPA. However, treatment with 50 or 100 mumol robinetin diminished ODC induction at 6 h after TPA. Treatment with 100 mumol apigenin or 50 or 100 mumol indole-3-carbinol in non-TPA-treated mouse skin caused elevations in epidermal ODC. In comparing the time course of ODC induction, indole-3-carbinol (50 mumol) pretreatment shifted the induction of epidermal ODC to earlier times, in addition to elevating ODC induction by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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