Dual responsive promoters to target therapeutic gene expression to radiation-resistant hypoxic tumor cells.
OBJECTIVE
Tumor hypoxia is unequivocally linked to poor radiotherapy outcome. This study aimed to identify enhancer sequences that respond maximally to a combination of radiation and hypoxia for use in genetic radiotherapy approaches.


METHODS
The influence of radiation (5 Gy) and hypoxia (1% O2) on reporter-gene expression driven by hypoxia (HRE) and radiation (Egr-1) responsive elements was evaluated in tumor cells grown as monolayers or multicellular spheroids. Hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha protein expression was monitored in parallel.


RESULTS
Of the sequences tested, an HRE from the phosphoglycerate kinase-1 gene (PGK-18[5+]) was maximally induced in response to hypoxia plus radiation in all 5 cell lines tested. The additional radiation treatment afforded a significant increase in the induction of PGK-18[5+] compared with hypoxia alone in 3 cell lines. HIF-1alpha/2alpha were induced by radiation but combined hypoxia/radiation treatment did not yield a further increase. The dual responsive nature of HREs was maintained when spheroids were irradiated after delivery of HRE constructs in a replication-deficient adenovirus.


CONCLUSIONS
Hypoxia-responsive enhancer element sequences are dually responsive to combined radiation and hypoxic treatment. Their use in genetic radiotherapy in vivo could maximize expression in the most radio-resistant population at the time of radiation and also exploit microenvironmental changes after radiotherapy to yield additional switch-on.
Chadderton
Naomi
N
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom.
Cowen
Rachel L
RL
Sheppard
Freda C D
FC
Robinson
Suzanne
S
Greco
Olga
O
Scott
Simon D
SD
Stratford
Ian J
IJ
Patterson
Adam V
AV
Williams
Kaye J
KJ
D051792:Basic Helix-Loop-Helix Proteins; D015687:Cell Hypoxia / Q000235:genetics*; D005786:Gene Expression Regulation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011836:Radiation Tolerance / Q000235:genetics* / Q000502:physiology; D018874:Spheroids, Cellular; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010735:Phosphoglycerate Kinase; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1