A novel chimeric promoter that is highly responsive to hypoxia and metals

• Published in: Gene therapy Vol. 13; no. 10; pp. 857 - 868
• Main Authors: LEE, J.-Y, LEE, Y.-S, KIM, D.-K, KIM, J.-M, KIM, K. L, LEE, J.-S, JANG, H.-S, SHIN, I.-S, SUH, W, JEON, E.-S, BYUN, J
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01.05.2006
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Binding sites; Biological and medical sciences; Biotechnology; Cancer; Carbon dioxide; Care and treatment; Cell Line; Cell Line, Tumor; Chimera; Chronic diseases; Cobalt; Cytomegalovirus; Deferoxamine; DNA, Single-Stranded - genetics; DNA-Binding Proteins - genetics; EGR-1 protein; Electrophoretic Mobility Shift Assay - methods; Enzyme-Linked Immunosorbent Assay - methods; Erythropoietin; Fundamental and applied biological sciences. Psychology; Gene expression; Gene therapy; Genetic aspects; Genetic Engineering; Health aspects; Health. Pharmaceutical industry; HeLa Cells; Hindlimb - blood supply; Human cytomegalovirus; Humans; Hydrogen peroxide; Hypoxia; Hypoxia - metabolism; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-inducible factor 1a; Hypoxia-inducible factors; Industrial applications and implications. Economical aspects; Ischemia; Kinases; Laser-Doppler Flowmetry; Medical sciences; Metallothionein; Metallothionein - genetics; Metals; Metals - metabolism; Mice; Molecular and cellular biology; Molecular genetics; Phosphoglycerate kinase; Phosphoglycerate Kinase - genetics; Phosphoglycerate kinase 1; Physiological aspects; Promoter Regions, Genetic; Promoters (Genetics); Regional Blood Flow; Regulatory sequences; Risk factors; Transcription Factor MTF-1; Transcription Factors - genetics; Transcription. Transcription factor. Splicing. Rna processing; Transfection; Transfection - methods; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; South Korea; MRE; Oxygen; Transcription promoter; inducibility; EBS; Hypoxia; HRE; promoter; Metal; Gene therapy
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3302728

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.