Development of synthetic promoters for radiation-mediated gene therapy.
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 adenocarcinoma 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 synthetic 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 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 herpes simplex virus thymidine kinase gene, leading to enhanced cell killing in the presence of the prodrug ganciclovir (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 transcriptional enhancers outside their normal sequence context. 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.
Marples
B
B
Cancer Research Campaign Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Manchester, UK.
Scott
S D
SD
Hendry
J H
JH
Embleton
M J
MJ
Lashford
L S
LS
Margison
G P
GP
D000230:Adenocarcinoma / Q000532:radiotherapy / Q000628:therapy*; D001943:Breast Neoplasms / Q000532:radiotherapy / Q000628:therapy*; D005260:Female; D015774:Ganciclovir / Q000627:therapeutic use; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005909:Glioblastoma / Q000532:radiotherapy / Q000628:therapy; D006801:Humans; D011401:Promoter Regions, Genetic; D011838:Radiation-Sensitizing Agents; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D011838:Radiation-Sensitizing Agents; D013937:Thymidine Kinase; D015774:Ganciclovir