Targeting diphtheria toxin A-chain transcription to activated endothelial cells using an E-selectin promoter.
Targeting the transcription of a toxin gene to activated endothelial cells might be used for inhibiting angiogenesis in solid tumors. As a model, we transiently transfected human endothelial cells (HUVEC) in culture with expression plasmids for the toxic A-chain of diphtheria toxin (DT-A), using electroporation (achieving approximately 70% transfection efficiency). Protein synthesis in HUVEC was highly sensitive to DT-A expression from constitutive viral promoters. E-selectin is strongly expressed on HUVEC activated by TNFalpha or TPA. We therefore tested a human E-selectin promoter (-547 to +33) for targeting transcription of DT-A or reporter genes to HUVEC. Luciferase reporters were efficiently expressed in HUVEC from this promoter, with or without an enhancer responsive to Ets-1. Expression was increased by TNF alpha or TPA. DT-A showed highly preferential expression (increased by TNF alpha or TPA) in HUVEC, compared with WI38 human fibroblasts. HUVEC expressing DT-A were killed via apoptosis. Overall expression levels were influenced by alternative 'backbone' sequences used in the expression plasmids. We propose that delivery of transcriptionally regulated expression plasmids for DT-A in vivo, using cationic lipids that show preferential accumulation in activated or proliferating endothelium, may offer a novel means of inhibiting undesired angiogenesis.
D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics*; D019040:E-Selectin / Q000235:genetics*; D042783:Endothelial Cells / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection
D004167:Diphtheria Toxin; D019040:E-Selectin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A