Abstract
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.
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References
Fornace AJ Jr et al. DNA damage inducible transcripts in mammalian cells Proc Natl Acad Sci USA 1988 85: 8800–8804
Woloschak GE et al. Low doses of neutrons induce changes in gene expression Radiat Res 1994 138: S56–S59
Sherman ML et al. Ionizing radiation regulates expression of the c-jun protooncogene Proc Natl Acad Sci USA 1990 87: 5663–5666
Hallahan DE et al. Increased tumor necrosis factor alpha mRNA after cellular exposure to ionizing radiation Proc Natl Acad Sci USA 1989 86: 10104–10107
Hallahan DE et al. Protein kinase C mediates X-ray inducibility of nuclear signal transducers EGR1 and Jun Proc Natl Acad Sci USA 1991 88: 2156–2160
Weichselbaum RR et al. Radiation induction of immediate early genes: effectors of the radiation stress response Int J Radiat Oncol Biol Phys 1994 30: 229–234
Boothman DA et al. Immediate X-ray inducible responses from mammalian cells Radiat Res 1994 138: S44–S46
Boothman DA et al. Isolation of an X-ray responsive element in the promoter region of tissue type plasminogen activator: potential uses of X-ray responsive elements for gene therapy Radiat Res 1994 138: S68–S71
Datta R et al. Ionizing radiation activates transcription of the EGR1 gene via CArG elements Proc Natl Acad Sci USA 1992 89: 10149–10153
Datta R et al. Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation Proc Natl Acad Sci USA 1993 90: 2419–2422
Joki T et al. Activation of the radiosensitive EGR-1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir Hum Gene Ther 1995 6: 1507–1513
Takahashi T et al. Induction of the suicide HSV TK gene by activation of the Egr 1 promoter with radioisotopes Hum Gene Ther 1997 8: 827–833
Gius D et al. Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate early gene expression via CArG elements Mol Cell Biol 1990 10: 4243–4255
Hallahan DE et al. Spatial and temporal control of gene therapy using ionizing radiation Nature Med 1995 1: 786–791
Mauceri HJ et al. Tumor necrosis factor alpha (TNF-alpha) gene therapy targeted by ionizing radiation selectively damages tumor vasculature Cancer Res 1996 56: 4311–4314
Seung LP et al. Genetic radiotherapy overcomes tumor resistance to cytotoxic agents Cancer Res 1995 55: 5561–5565
Sakamoto KM et al. 5′ upstream sequence and genomic structure of the human primary response gene, EGR-1/TIS8 Oncogene 1991 6: 867–871
Mauceri HJ et al. Increased injection number enhances adenoviral genetic radiotherapy Radiat Oncol Invest 1997 5: 220–226
Mensil M et al. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins Proc Natl Acad Sci USA 1996 93: 1831–1835
Balzarini J et al. Comparative cytostatic activity of different antiherpertic drugs against herpes simplex virus thymidine kinase gene-transfected tumour cells Mol Pharm 1994 45: 1253–1258
Degreve B et al. Bystander effect of purine nucleoside analogues in HSV-1tk suicide gene therapy is superior to that of pyrimidine nucleoside analogues Gene Therapy 1999 6: 162–170
Stevens CW et al. Ionizing radiation greatly improves gene transfer efficiency in mammalian cells Hum Gene Ther 1996 7: 1727–1734
Pederson LC et al. Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo Cancer Res 1997 57: 4325–4332
Weichselbaum RR et al. Gene therapy targeted by ionizing radiation Int J Radiat Oncol Biol Phys 1992 24: 565–567
McBride WH, Dougherty GJ . Radiotherapy for genes that cause cancer Nature Med 1995 1: 1215–1217
Wagner MJ, Sharp JA, Summers WC . Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1 Proc Natl Acad Sci USA 1981 78: 1441–1445
Acknowledgements
This work was supported by the Friends of Rosie Children's Cancer Research Fund, Christie Hospital NHS Trust Endowment Fund and The Cancer Research Campaign. The authors thank Dr Stephen Roberts for statistical analysis and Rachel Williams and Rachel Exley for technical assistance.
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Marples, B., Scott, S., Hendry, J. et al. Development of synthetic promoters for radiation-mediated gene therapy. Gene Ther 7, 511–517 (2000). https://doi.org/10.1038/sj.gt.3301116
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DOI: https://doi.org/10.1038/sj.gt.3301116
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