1. ¹Tumour Targeting Group, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK
2. ²Cancer Research UK, Molecular Oncology Laboratory, Institute of Molecular Medicine, University of Oxford, Headington, Oxford OX3 9DU, UK

Correspondence: Professor C Lewis, E-mail: Claire.lewis@sheffield.ac.uk

³Current address: Department of Microbiology & Immunology, University of Leicester, Leicester LE1 9HN, UK

Received 21 March 2002; Revised 5 August 2002; Accepted 15 August 2002.

Abstract

Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements. The activity of such elements in hypoxic cells is directly dependent on upregulation of the hypoxia-inducible transcription factor-1 However tumours also contain areas of anoxia, which may be considered a more tumour-selective transcriptional stimulus than hypoxia for targeting gene therapy to tumours. Another element, from the rat virus-like retrotransposon, VL30 (termed the 'secondary anoxia response element') has been reported to be more highly inducible in rat fibroblasts under anoxia than hypoxia. To investigate anoxia as a potential transcriptional target in human tumours, we have examined secondary anoxia response element inducibility in two human breast cancer cell lines, MCF-7 and T47D, under anoxia, hypoxia and normoxia. In both cell types, the trimerised secondary anoxia response element showed greater inducibility in anoxia than hypoxia (1% and 0.5% O₂). The anoxic response of the secondary anoxia response element was shown to be dependent on hypoxia-inducible transcription factor-1 and the presence of a hypoxia-inducible transcription binding site consensus (5'-ACGTG-3'). Mutational analysis demonstrated that the base immediately 5' to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>>CACGTG. A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5' flanking bases.