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

Vincent Fong,Christopher Hine,Marika Osterbur,Vera Gorbunova,Stephen Dewhurst,Yo-El Kim,Andrei Seluanov,Cristina Capella

doi:10.1371/journal.pone.0028714

Abstract

BackgroundThe 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 FindingsExpression 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/SignificanceOverall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53.

Highlights

Specific targeting of therapeutic agents to cancer cells while avoiding damage to normal tissue has been a long time goal in cancer research
In order to better assess the differential expression of the Rad51promoter, we generated a panel of truncated Rad51 promoter mutants (Figure 1), inserted them upstream of a promoterless luciferase reporter and produced a series of replication-defective, E1-deleted Ad5 vectors that were evaluated in a panel of normal and cancer cell lines (Table 1)
Luciferase activity in cells transduced with a vector containing this element (Rad51core-luc) was essentially indistinguishable from that in cells transduced with vectors containing larger fragments of the Rad51 promoter (Rad51-F6luc, 22931/+217; Rad51-D230-luc, 2230/+3564) or the intact full-length Rad51 promoter (22931/+3564) (Figure 2A)

Summary

Introduction

Specific targeting of therapeutic agents to cancer cells while avoiding damage to normal tissue has been a long time goal in cancer research. One method of targeting viral agents has been to use tumor specific promoters to restrict expression of therapeutic genes [1,2]. Gorbunova and colleagues reported that the full length Rad promoter maintains its cancer specificity when taken independent of its natural context and showed that it can drive tumor-selective expression of a reporter gene [18]. This makes the Rad promoter a very attractive candidate for use in anti-cancer therapies especially when coupled with the efficient transduction capabilities of viral vectors [19]. We set out to assess whether we could confer this tumor-selectivity to an adenovirus vector

Methods

Results

Conclusion