| Objective A novel approach, exploiting hypoxic and radiation-response elements within a single gene promoter to control expression of a therapeutic gene, would restrict therapeutic gene activation to hypoxic and/or irradiated tissues. Since gene activation would be targeted, damage to normal tissue surrounding the tumor would be reduced, thereby, the therapeutic ratio would be improved. The aim of this study was to develop therapeutic strategies for lung cancer by constructing chimeric gene promoters containing hypoxia regulatory elements (HREs) and radiation- responsive promoter from the early growth response (Egr) 1 gene, known as CArG elements, which responsive to hypoxia and ionizing radiation.Materials and methods The pcDNA3.1(-) plasmid was used as the basis for all new constructs. Adopting molecular cloning methods, we constructed the plasmids of pDNA.miniCMV.EGFP , pDNA.HRE.miniCMV.EGFP, pDNA. CArG.miniCMV.EGFP, pDNA.HRE.CArG.miniCMV.EGFP, pDNA.miniCMV. HSVtk, pDNA.HRE.miniCMV.HSVtk , pDNA.CArG.miniCMV.HSVtk and pDNA.HRE.CArG.miniCMV.HSVtk.Results pcDNA3.1(-) plasmid was excised by Nru I /EcoR V digestion,then one ligation was reacted. The new plasmid was produced, named as pDNA plasmid. The pDNA plasmid was excised by Acc65I/HindIII digestion, the miniCMV promoter, containing one Xhol site, was then cloned into pDNA plasmid backbone between Acc65I and HindIII sites in one ligation reaction, producing a new plasmid named as pDNA.miniCMV. The pDNA.miniCMV plasmid was excised by Mfel/ BamHI sites. The HRE units were cloned into pDNA.miniCMV backbone in one ligation reaction. The new plasmid was called pDNA.HRE.miniCMV. The CArG elements were cloned into pDNA. miniCMV or pDNA.HRE.miniCMV backbone between BamHI and Acc65I sites in one ligation reaction, adjacently and immediately upstream of miniCMV promoter. The new plasmids were called pDNA.CArG.miniCMV and pDNA. HRE.CArG.miniCMV. The EGFP or HSVtk gene was inserted into these plasmids between Xhol and Hindlll sites. The sequence integrity of all plasmids was confirmed by Shanghai Biological Engineering Technology and Service Company.Conclusion The chimeric gene promoters containing hypoxic and radiation-response elements to control expression of a therapeutic gene were constructed.Part twoChimeric Gene Promoters Responsive to Hypoxia and Ionizing Radiation to Control the Expression of EGFP GeneObjective To observe the expression of the enhanced green fluorescent protein (EGFP) induced by chimeric gene promoters containing hypoxic and radiation- response elements in SPCA1 and A549 non-small-cell lung cancer (NSCLC) cells under the circumstance of hypoxia and/or ionizing radiation.Materials and methods Transient transfectants with the EGFP-constructs were obtained by exposing the SPCA1 and A549 cells to complexes of DNA, polyethylenimine (PEI) for efficient transfection. The cells were exposed to hypoxia and/or radiation 24 h after transfection. Relative EGFP fluorescence was measured by fluorescence activated cell-sorting (FACS) analysis. The level of hypoxia- and/or radiation- induced EGFP expression was expressed as the percentage of EGFP- expressing cells in the treated sample, compared with the corresponding oxic/sham-irradiated sample transfected with the same plasmid.Results Plasmid transfection efficiencies were 31.2±3.2% in SPCA1 and 27.8±2.7% for A549 cells. Compared with the corresponding oxic/sham -irradiated sample transfected with the same plasmid, The fold induction of EGFP expression levels in radiation SPCA1 and A549 cells transiently transfected with the pDNA.HRE.miniCMV.EGFP plasmid were 1.85±0.18 and 2.14±0.25-fold, in hypoxic SPCA1 and A549 cells were 2.02±0.23 and 2.37±0.24-fold, in hypoxia and radiation SPCA1 and A549 cells were 2.39±0.26 and 2.35±0.34-fold, respectively. The fold induction of EGFP expression levels in radiation SPCA1 and A549 cells transiently transfected with the pDNA.CArG.miniCMV.EGFP plasmid were 2.56±0.21 and 2.81±0.31 -fold, inhypoxic SPCAl and A549 cells were 1.84±0.12 and 2.07±0.18-fold, in hypoxia and radiation SPCAl and A549 cells were 2.18±0.21 and 1.83±0.15-fold, respectively. The fold induction of EGFP expression levels in radiation SPCAl and A549 cells transiently transfected with the pDNA.HRE.CArG.miniCMV. EGFP plasmid were 2.69±0.14 and 3.16±0.27-fold, in hypoxic SPCAl and A549 cells were 2.41±0.18 and 2.89±0.25-fold, in hypoxia and radiation SPCAl and A549 cells were 3.37±0.23 and 3.10±0.28-fold, respectively. The CMV or miniCMV promoter showed no induction in either cell type.Conclusion The data indicate that the chimeric promoters containing hypoxic and radiation-response elements were able to respond to hypoxia and radiation. The pDNA.HRE.CArG.miniCMV.EGFP plasmid proved to be themost responsive element combination.Part threeChimeric Gene Promoters to Control the Expression of HSVtk Gene toKill Lung Cancer CellsObjective To examine the potential of chimeric promoters responsive to hypoxia and ionizing radiation to control the expression of HSVtk- mediated gene-directed enzyme prodrug therapy (GDEPT) to kill lung cancer cells.Materials and methods Transient transfectants with the HSVtk- constructs were obtained by exposing the SPCAl and A549 cells to complexes of DNA, polyethylenimine (PEI) for efficient transfection. Following hypoxic and/or radiation exposure, cells were exposed to media containing ganciclovir (GCV)50 uM for 24 h. Two days later, tumor cell growth were determined by [3-(4,5-dimcthylthioazol- 2-yl)-2,5-diphenyl-tetrazolium bromide, MTT] growth delay assay.Results Compared with the corresponding oxic/sham -irradiated sample transfected with the same plasmid, cell survival of SPCA1 and A549 cells transiently transfected with the pDNA.HRE.miniCMV.HSVtk plasmid in radiation and GCV exposure group reduced 26.54±1.77% and 34.12±1.48%, in hypoxic and GCV exposure group reduced 29.9±1.60% and 42.83±2.57%, in hypoxia and radiation and GCV exposure group reduced 39.73±2.00% and 55.73±2.22%, respectively; cell survival of SPCA1 and A549 cells transiently transfected with the pDNA.CArG.miniCMV.HSVtk plasmid in radiation and GCV exposure group reduced 32.08±2.43% and 42.87±1.61%, in hypoxic and GCV exposure group reduced 19.14±1.36% and 31.01±1.59%, in hypoxia and radiation and GCV exposure group reduced 39.73±2.00% and 55.73±2.22%, respectively; cell survival of SPCA1 and A549 cells transiently transfected with the pDNA.HRE.CArG.miniCMV.HSVtk plasmid in radiation and GCV exposure group reduced 41.59±1.38% and 49.06±3.05%, in hypoxic and GCV exposure group reduced 39.37±1.25% and 50.99±1.37%, in hypoxia and radiation and GCV exposure group reduced 63.23±2.31% and 76.58±2.19%, respectively.Conclusion The data indicate that the chimeric promoters containing hypoxic and radiation- response elements we constructed were respond to hypoxia and radiation. The addition of GCV to the irradiation and/or hypoxia treatment significantly reduced plasmid-transfected cells survival. The pDNA.HRE.CArG.miniCMV.HSVtk plasmid proved to be the most responsive...
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