Gene Therapy (2000) 7, 511–517  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt ACQUIRED DISEASES RESEARCH ARTICLE Development of synthetic promoters for radiation- mediated gene therapy B Marples 1,4 , SD Scott 1,4 , JH Hendry 1 , MJ Embleton 2 , LS Lashford 3 and GP Margison 1 Cancer Research Campaign Sections of 1 Genome Damage and Repair, 2 Cell and Tumour Biology, and 3 Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, and 3 Academic Department of Paediatric Oncology, Christie Hospital (NHS) Trust, Manchester, UK Exposure of cells to ionising radiation results in the activation of specific transcriptional control (CArG) elements within the early growth response 1 (Egr1) gene promoter, leading to increased gene expression. As part of a study investigating the potential use of these elements in radiation-controlled gene therapy vectors, we have incorporated their sequences into a synthetic gene promoter and assayed for the ability to induce expression of a downstream reporter gene following irradiation. In vector-transfected MCF-7 breast adenocarcin- oma cells, the synthetic promoter was more effective than the wild-type Egr1 counterpart in up-regulating expression of the reporter gene after exposure to a single 5 Gy dose, and equally effective as the wild-type in U87-MG glioma cells. The level of gene expression achieved using the syn- thetic promoter was dependent on the inducing radiation dose for both U87-MG and MCF-7 cells, being maximal at 3 Gy and decreasing at 5 and 10 Gy. Furthermore, induction Keywords: Egr1; CArG elements; GFP; gene therapy Introduction The expression of some early response genes is up-regu- lated by exposure to ionising radiation. 1–7 These genes include those encoding the human tissue-type plasmin- ogen activator, tumour necrosis factor alpha (TNF-) and early growth response protein 1 (Egr1). 8,9 Transcriptional activation of the Egr1 gene by ionising radiation appears to be a direct consequence of the production of reactive oxygen intermediates. 10 Although the precise activation mechanism has not been fully elucidated, 10 base pair sequences known as CArG elements have been identified as radio-responsive motifs within the Egr1 promoter, the majority of which are positioned within serum response elements. 9,10 However, only the CArG elements grouped in the 5' distal ‘enhancer’ region, appear to contribute to the radiation responsiveness of Egr1. 9 Correspondence: B Marples, Experimental Oncology, Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, UK 4 Present address: Experimental Oncology, Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, UK Received 24 April 1998; revised 9 August 1999; accepted 8 Nov- ember 1999 could be repeated by additional radiation treatments. The latter indicates that up-regulation should be additive during fractionated radiotherapy schedules. To demonstrate the potential clinical benefit of such an approach, the synthetic promoters were also shown to drive expression of the her- pes simplex virus thymidine kinase gene, leading to enhanced cell killing in the presence of the prodrug ganciclo- vir (GCV) when compared with cells treated with radiation alone. Our results demonstrate that the synthetic promoter is responsive to low doses of ionising radiation and therefore isolated CArG elements function as radiation-mediated tran- scriptional enhancers outside their normal sequence con- text. The continued development and optimisation of such radiation-responsive synthetic promoters is expected to make a valuable contribution to the development of future radiation-responsive vectors for cancer gene therapy. Gene Therapy (2000) 7, 511–517. The radiation inducibility of the Egr1 promoter has been exploited in several experimental gene therapy stra- tegies. Joki et al 11 and Takahashi et al 12 used a plasmid construct containing Egr1 gene sequences (positions -425 to +65 within the wild-type promoter) containing six CArG elements (first described in Gius et al 13 ), in combi- nation with the herpes simplex virus thymidine kinase (HSVtk) gene. Glioma cells containing this vector were sensitised to the antiviral agent ganciclovir (GCV), a sub- strate for HSVtk, after exposure to a single radiation dose of 20 Gy. 11 Direct intra-tumoural injection has also been used to deliver an adenovirus vector containing Egr1 enhancer-controlled TNF- gene to human epithelia SQ- 20B tumour xenografts. 14,15 In these studies a radiation dose schedule of 40 Gy, given as 5 Gy fractions twice a day for 4 days, increased tumour regression by up to 90% of control values compared with maxima of 50% for vector or radiation alone. 14 Similar results were obtained using a murine fibrosarcoma model. 16 To date, there has been no evidence as to whether CArG elements will function independently of their natu- ral sequence context, in which other potential regulatory regions such as putative binding sites for the transcrip- tion factors SP1 and AP-1 (tetra-decanoyl phorbol acetate or TPA responsive element), and the cAMP-responsive element binding protein are positioned. 17 In this study,