Richard Iggo

We have used chromatin immunoprecipitation (ChIP) to measure p53-dependent histone acetylation at the p21, MDM2, and PUMA promoters. The pattern of histone acetylation was different at each promoter. H3 and H4 acetylation increased at both the p21 and PUMA promoters in response to p53 activation, whereas there was only a minimal increase in H4 acetylation and no increase in H3 acetylation at the MDM2 promoter. The high p53 occupancy of the p21, MDM2 and PUMA promoters has been attributed to the presence of two p53 binding sites in these promoters, but mutation of the p53 binding sites in integrated p21 promoter constructs showed that the two sites in the p21 promoter do not cooperate to stabilize p53 binding. Despite 10-fold higher p53 binding to the proximal than the distal site in the p21 promoter, both sites showed similar patterns of H3 and H4 acetylation. Mutation of the binding sites showed that acetylation of the proximal, low-affinity site requires p53 binding to that site but not to the distal, high-affinity site. Since low-affinity p53 binding sites can confer strong acetylation, the DNA binding affinity in vitro is an unreliable guide to the likely importance of p53 in regulating candidate target genes in vivo.

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and β-catenin genes. We have modified the minute virus of mice (MVM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric β-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of ΔN-β-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy.

P53 can adopt two forms in vitro , a latent form that binds naked DNA poorly and an active form that binds DNA well. Conversion of the latent form to the active form is thought to occur by an allosteric mechanism induced by phosphorylation and acetylation. Despite the large differences in affinity produced by regulatory modifications in vitro , mutation of putative regulatory sites has not produced correspondingly large effects on transcription of p53 target genes in vivo . To determine whether genotoxic stress regulates DNA binding by p53 in vivo , we have performed quantitative chromatin immunoprecipitation (ChIP) assays on tumor and normal cell lines containing wild-type p53. ChIP recovers several hundredfold more p21 and MDM2 promoter DNA from p53 wild-type than p53-null cells, indicating that the assay is specific for p53. Genotoxic stress induces much smaller increases in chromatin precipitation, which are matched by changes in the p53 protein level. Thus, in the experimental systems tested, allosteric regulation of DNA binding is not a major level of regulation of p53 activity. The p53 target genes tested can be divided into a group showing high promoter occupancy in vivo (p21, MDM2, and PUMA) and a group giving substantially weaker or background p53 binding (bax, AIP1, and PIG3). Neither group shows selective recruitment of p53 to the promoter in cells undergoing apoptosis, indicating that the decision to undergo apoptosis or cell cycle arrest depends on other changes in the cell.

Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and β-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable β-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.

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