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.
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.
Koshikawa
Nobuko
N
Division of Chemotherapy, Chiba Cancer Center Research Institute, Japan.
Takenaga
Keizo
K
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
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