Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.
We have been developing synthetic gene promoters responsive to clinical doses of ionizing radiation (IR) for use in suicide gene therapy vectors. The crucial DNA sequences utilized are units with the consensus motif CC(A/T)(6)GG, known as CArG elements, derived from the IR-responsive Egr1 gene. In this study we have investigated the parameters needed to enhance promoter activation to radiation. A series of plasmid vectors containing different enhancer/promoters were constructed, transiently transfected into tumor cells (MCF-7 breast adenocarcinoma and U-373MG glioblastoma) and expression of a downstream reporter assayed. Results revealed that increasing the number of CArG elements, up to a certain level, increased promoter radiation-response; from a fold-induction of 1.95 +/- 0.17 for four elements to 2.74 +/- 0.17 for nine CArGs of the same sequence (for MCF-7 cells). Specific alteration of the core A/T sequences caused an even greater positive response, with fold-inductions of 1.71 +/- 0.23 for six elements of prototype sequence compared with 2.96 +/- 0.52 for one of the new sequences following irradiation. Alteration of spacing (from six to 18 nucleotides) between elements had little effect, as did the addition of an adjacent Sp1 binding site. Combining the optimum number and sequence of CArG elements in an additional enhancer was found to produce the best IR induction levels. Furthermore, the improved enhancers also performed better than the previously reported prototype when used in in vitro and in vivo experimental GDEPT. We envisage such enhancers will be used to drive suicide gene expression from vectors delivered to a tumor within an irradiated field. The modest, but tight expression described in the present study could be amplified using a molecular 'switch' system as previously described using Cre/LoxP. In combination with targeted delivery, this strategy has great potential for significantly improving the efficacy of cancer treatment in the large number of cases where radiotherapy is currently employed.
D000230:Adenocarcinoma / Q000628:therapy; D000818:Animals; D001943:Breast Neoplasms / Q000628:therapy; D004742:Enhancer Elements, Genetic; D004792:Enzyme Precursors / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005909:Glioblastoma / Q000628:therapy; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000532:radiotherapy / Q000628:therapy*; D009374:Neoplasms, Experimental / Q000628:therapy; D011401:Promoter Regions, Genetic; D011878:Radiotherapy / Q000379:methods*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors