Gene Therapy (2002) 9, 263–269  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Tumour cell radiosensitization using constitutive (CMV) and radiation inducible (WAF1) promoters to drive the iNOS gene: a novel suicide gene therapy J Worthington, T Robson, M O’Keeffe and DG Hirst Radiation Science Research Group, School of Biomedical Sciences, University of Ulster, Newtownabbey, Co. Antrim, UK Nitric oxide (NO  ) has many characteristics including cyto- toxicity, radiosensitization and anti-angiogenesis, which make it an attractive molecule for use in cancer therapy. We have investigated the use of iNOS gene transfer, driven by both a constitutive (CMV) and X-ray inducible (WAF1) pro- moter, for generating high concentrations of NO  within tumour cells. We have combined this treatment with radi- ation to exploit the radiosensitizing properties of this mol- ecule. Transfection of murine RIF-1 tumour cells in vitro with the iNOS constructs resulted in increased iNOS protein lev- els. Under hypoxic conditions cells were radiosensitized by delivery of both constructs so that these treatments effec- Keywords: radiosensitization; inducible nitric oxide synthase; cancer gene therapy Introduction Nitric oxide (NO  ) is an important intra- and inter-cellu- lar mediator, governing a plethora of physiological func- tions, from the control of smooth muscle tone in the cardiovascular, gastrointestinal, respiratory and genito- urinary systems, to neurotransmission. 1 It is also involved in immune function and inflammation. 2 The role of NO  in tumour biology is complex – it is involved in regulating tumour cell growth and proliferation, it affects transcription of certain genes, and plays a role in host response to tumour growth. 3 NO  production in solid tumours has been implicated in enhanced vascular permeability and increased tumour blood flow; 4 inhi- bition of NO  synthase (NOS) has been shown to retard tumour growth in vivo. 5 However, the level of NO  appears to be critical in determining the tissue response. When NO  is generated at high concentrations, such as by macrophages, it can cause cell death in tumours. 6 It has also been shown that NO  -donor molecules can inhibit vessel formation in the chick chorioallontoic mem- brane and reverse the angiogenic effect of alpha thrombin. 7 NO  has been widely reported as an hypoxic cell radiosensitizer. 8–10 Using authentic NO  gas, Mitchell et al 8 demonstrated that NO  was almost as effective as oxy- Correspondence: DG Hirst, Radiation Science Research Group, School of Biomedical Sciences, University of Ulster, Newtownabbey, Co. Antrim, BT37 0QB Received 17 July 2001; accepted 29 October 2001 tively eliminated the radioresistance observed under hypoxic conditions. In vivo transfer of the CMV/iNOS construct by direct tumour injection resulted in a delay (4.2 days) in tumour growth compared with untreated controls. This was equivalent to the effect of 20 Gy X-rays alone. Combination of CMV/iNOS gene transfer with 20 Gy X-rays resulted in a dramatic 19.8 day growth delay compared with controls. Tumours treated with the CMV/iNOS showed large areas of necrosis and abundant apoptosis. We believe that iNOS gene transfer has the potential to be a highly effective treat- ment in combination with radiotherapy. Gene Therapy (2002) 9, 263–269. DOI: 10.1038/sj/gt/3301609 gen in sensitizing hypoxic Chinese hamster V79 lung cells to radiation, with a sensitizer enhancement ratio (SER) of 2.3 at a concentration of 10 4 p.p.m. In the same study the NO  donor 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) gave an SER of 2.9 at a concen- tration of 2 mm. Very similar results were reported for DEA/NO (SER = 3.0 at 2 mm) and for another NO  donor, spermine/nitric oxide complex (SER = 2.8 at 2 mm) in SCK tumour cells in vitro. 11 Kurimoto et al 10 exam- ined the radiosensitizing effect of the NO  -generating agents S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in glioma cells, observing SERs of 1.4 and 1.9 respectively at concentrations of 100 m. Furthermore, a rise in NO  levels as a secondary consequence of IFN- exposure in EMT-6 tumour cells resulted in hypoxic cell radiosensitization. 9 Spontaneous and bioreductive NO  donors, such as SNP and SNAP, elicit systemic vasodilation and hypoten- sion; they cannot therefore be administered in sufficient doses to achieve tumour radiosensitization in the clinic. One strategy for generating NO  specifically within the tumour is the use of iNOS gene therapy. The potential for iNOS in a suicide gene therapy approach has already been effectively demonstrated by several other groups, 12–16 but we are not aware of any studies in which iNOS gene transfer was combined with radiotherapy. We have previously demonstrated that transfection of rat arteries with the iNOS gene, under the control of a constitutive CMV promoter, can modify vascular tone by generation of large amounts of NO  . Furthermore, we have shown that we can achieve similar effects by using