Adenoviral Vector Driven by a Minimal Rad51 Promoter Is Selective for p53-Deficient Tumor Cells

Background: The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector. Methodology/Principal Findings: Expression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 null cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir. Conclusions/Significance: Overall, these experiments demonstrated that a small core domain of the Rad51 promoter ca

The possible functions of duplicated ets (GGAA) motifs located near transcription start sites of various human genes

Transcription is one of the most fundamental nuclear functions and is an enzyme complex-mediated reaction that converts DNA sequences into mRNA. Analyzing DNA sequences of 5′-flanking regions of several human genes that respond to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in HL-60 cells, we have identified that the ets (GGAA) motifs are duplicated, overlapped, or clustered within a 500-bp distance from the most 5′-upstream region of the cDNA. Multiple protein factors including Ets family proteins are known to recognize and bind to the GGAA containing sequences. In addition, it has been reported that the ets motifs play important roles in regulation of various promoters. Here, we propose a molecular mechanism, defined by the presence of duplication and multiplication of the GGAA motifs, that is responsible for the initiation of transcription of several genes and for the recruitment of binding proteins to the transcription start site (TSS) of TATA-less promoters

Multiple protein binding sites within the ovalbumin gene 5'-flanking region: isolation and characterization of sequence-specific binding proteins.

To understand the molecular basis of the steroid hormone regulated expression of the ovalbumin gene, we sought to identify and isolate nuclear factors from chicken oviduct which interact specifically with the ovalbumin promoter. Using DNase I footprinting and mobility shift assays, we have defined at least four distinct protein binding sites, OV-150, OV-220, OV-250 and OV-330, in the promoter region between -100 to -400. Binding competition and protein fractionation studies revealed the existence of two distinct proteins, each recognizing two promoter sites: Both OV-330 and OV-250 are recognized by one protein factor which is distinct from the one binding to both OV-220 and OV-150. The location of the DNase I footprints coincides with those of in vivo chromatin hypersensitive sites. The OV-330 site is located in a sequence area required for the repression of the gene in the absence of hormone. The factor binding to OV-330 has been substantially purified and renaturation experiments indicate that the binding activity is associated with a polypeptide(s) of Mr 40K