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

Gene Ther. 2006 May;13(10):857-68. doi: 10.1038/sj.gt.3302728.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Line, Tumor
  • Chimera
  • DNA, Single-Stranded / genetics
  • DNA-Binding Proteins / genetics
  • Electrophoretic Mobility Shift Assay / methods
  • Enzyme-Linked Immunosorbent Assay / methods
  • Genetic Engineering*
  • HeLa Cells
  • Hindlimb / blood supply
  • Humans
  • Hypoxia / metabolism*
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Laser-Doppler Flowmetry
  • Metallothionein / genetics
  • Metals / metabolism*
  • Mice
  • Phosphoglycerate Kinase / genetics
  • Promoter Regions, Genetic*
  • Regional Blood Flow
  • Transcription Factors / genetics
  • Transfection / methods

Substances

  • DNA enzyme ED5
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Metals
  • Transcription Factors
  • transcription factor MTF-1
  • Metallothionein
  • Phosphoglycerate Kinase