title: Hypoxia-regulated expression of attenuated diphtheria toxin A fused with hypoxia-inducible factor-1alpha oxygen-dependent degradation domain preferentially induces apoptosis of hypoxic cells in solid tumor.
abstract: Tumor cells in hypoxic areas of solid tumors are resistant to conventional chemotherapy and radiotherapy and thus are obstacles of cancer therapy. We report here the feasibility of applying hypoxia-regulated expression of diphtheria toxin A (DT-A) for killing hypoxic tumor cells. The expression vector was constructed to express DT-A fused with hypoxia-inducible factor-1alpha (HIF-1alpha) oxygen-dependent degradation (ODD) domain under the control of vascular endothelial growth factor gene promoter and contain erythropoietin mRNA-binding protein (ERBP)-binding sequence downstream of the DT-A/ODD sequence. In vitro ubiquitination assay showed that DT-A/ODD, but not DT-A, was ubiquitinated as efficient as HIF-1alpha under normoxic conditions in a von Hippel-Lindau- and oxygen-dependent manner. DT-A/ODD exhibited a comparable translation inhibitory activity to DT-A. ERBP-binding sequence was effective in stabilizing mRNA under hypoxic conditions in various cell types. Transfection of the vector expressing DT-A/ODD into high-metastatic Lewis lung carcinoma (3LL) A11 cells resulted in induction of apoptosis independently of hypoxia, probably due to its extreme toxicity. However, transfection of the vector expressing attenuated DT-A(W153F)/ODD or DT-A(H21A)/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-A(W153F)/ODD induced apoptosis in hypoxic, but not in normoxic, areas of solid tumors established by A11 variant cells with higher resistance to hypoxia-induced apoptosis and inhibited the growth of hypoxic tumors established by 3LL-P29 cells. These results suggest that hypoxia-regulated expression of attenuated DT-A(W153F)/ODD fusion protein is potentially of use for killing hypoxic tumor cells with minimizing the damage to normoxic normal tissues.
authors.affiliation: Division of Chemotherapy, Chiba Cancer Center Research Institute, Japan.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Koshikawa
authors.forename: Keizo
authors.initials: K
authors.lastname: Takenaga
mesh_terms: D000818:Animals; D017209:Apoptosis; D018827:Carcinoma, Lewis Lung / Q000378:metabolism / Q000473:pathology / Q000556:secondary; D015687:Cell Hypoxia; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D004921:Erythropoietin / Q000235:genetics; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000378:metabolism / Q000473:pathology; D008081:Liposomes; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger; D016601:RNA-Binding Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D004167:Diphtheria Toxin; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008081:Liposomes; D010446:Peptide Fragments; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D042461:Vascular Endothelial Growth Factor A; C029795:diphtheria toxin fragment A; D004921:Erythropoietin; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D010100:Oxygen