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Hypoxia-Regulated Expression of Attenuated Diphtheria Toxin A Fused with Hypoxia-Inducible Factor-1 Oxygen-Dependent Degradation Domain Preferentially Induces Apoptosis of Hypoxic Cells in Solid Tumor

Divisions of ¹ Chemotherapy and ² Pathology, Chiba Cancer Center Research Institute, Chiba, Japan

Requests for reprints: Keizo Takenaga, Division of Chemotherapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuoh-ku, 260-8717 Chiba, Japan. Phone: 81-43-264-5431; Fax: 81-43-265-4459; E-mail: keizo{at}chiba-cc.jp.

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-1 (HIF-1) 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-1 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. (Cancer Res 2005; 65(24): 11622-30)