Use of the radiation-inducible WAF1 promoter to drive iNOS gene therapy as a novel anti-cancer treatment.
BACKGROUND
Inducible nitric oxide synthase (iNOS) gene therapy has been identified as a potential anti-tumour strategy. A major problem common to most gene therapy strategies is targeting of treatment to the tumour volume. In this study we report on the use of the X-ray-inducible WAF1 promoter to achieve targeting of iNOS expression to the tumour volume.


METHODS
A WAF1/iNOS/liposome complex was injected directly into RIF-1 and HT29 tumours in mice. A 4 Gy dose of X-rays was applied to induce the WAF1 promoter followed, 8 h later, by treatment doses of 10 or 20 Gy. Tumour volume was measured, and growth curves plotted.


RESULTS
Intra-tumoural injection of WAF1/iNOS combined with a priming dose of X-rays to induce the WAF1 promoter, followed by a treatment dose, resulted in sensitiser enhancement ratios of 2.0 and 1.3 in RIF-1 and HT29 tumours, respectively, compared with radiation treatment alone. PCR analysis of organ tissue after intra-tumoural injection of WAF1/iNOS showed that vector sequences were detected in all tissue tested; however, Western blot analysis revealed that iNOS protein levels were significantly increased only in tumour and the surrounding dermal tissue that had been exposed to the 4 Gy inducing dose.


CONCLUSIONS
iNOS gene therapy in combination with an inducible promoter results in significant tumour cell radiosensitisation. The WAF1 promoter may be a good candidate for a gene therapy as it is silent in normal tissue yet can be induced by the tumour environment.
Worthington
Jenny
J
Radiation Science Group, School of Biomedical Sciences, University of Ulster, Shore Road, Newtownabbey, Co. Antrim, BT37 0QB, UK. J.Worthington@ulster.ac.uk
McCarthy
Helen O
HO
Barrett
Eimear
E
Adams
Catherine
C
Robson
Tracy
T
Hirst
David G
DG
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000528:radiation effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000493:pharmacokinetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009369:Neoplasms / Q000235:genetics / Q000628:therapy*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000528:radiation effects*; D014018:Tissue Distribution; D014965:X-Rays; D023041:Xenograft Model Antitumor Assays
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse