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Antitumor Effect of TAT-Oxygen-Dependent Degradation-Caspase-3 Fusion Protein Specifically Stabilized and Activated in Hypoxic Tumor Cells

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Antitumor Effect of TAT-Oxygen-Dependent Degradation-Caspase-3 Fusion Protein Specifically Stabilized and Activated in Hypoxic Tumor Cells [CANCER RESEARCH 62, 2013–2018, April 1, 2002] Antitumor Effect of TAT-Oxygen-dependent Degradation-Caspase-3 Fusion Protein Specifically Stabilized and Activated in Hypoxic Tumor Cells Hiroshi Harada, Masahiro Hiraoka, and Shinae Kizaka-Kondoh1 Pharmaceuticals Division, Pharmaceutical R&D Department, POLA Chemical Industries, Inc., Totsuka-ku, Yokohama 244-0812 [H. H.]; Department of Therapeutic Radiology and Oncology, Kyoto University Graduate School of Medicine, Shogoin, Sakyo-ku, Kyoto 606-8507 [M. H.]; and Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501 [S. K-K.], Japan ABSTRACT The ODD domain of HIF-1␣ is located in its central region and consists of ϳ200 amino acid residues. The ODD domain controls the Human solid tumors contain hypoxic regions that have considerably degradation of HIF-1␣ by the ubiquitin-proteasome pathway, and the lower oxygen tension than normal tissues. These impart resistance to deletion of this entire region is required for DNA binding and trans- radiotherapy and anticancer chemotherapy, as well as predisposing to increased tumor metastases. To develop a potentially therapeutic protein activation in the absence of hypoxic signaling (15). Recently, the drug highly specific for solid tumors, we constructed fusion proteins product of von Hippel-Lindau tumor suppressor gene was reported to selectively stabilized in hypoxic tumor cells. A model fusion protein, mediate ubiquitination and proteasomal degradation of HIF-1␣ under oxygen-dependent degradation (ODD)-␤-galactosidase (␤-Gal), composed normoxic conditions by interaction with the core of the ODD domain of a part of the ODD domain of hypoxia-inducible factor-1␣ fused to of HIF-1␣ (16–18). ␤-Gal, showed increased stability in cultured cells under a hypoxia-mimic HIV-1 TAT has been used to deliver functional biomolecules into condition. When ODD-␤-Gal was further fused to the HIV-TAT protein cells. Although the entire protein can be used for this purpose, it is transduction domain (TAT47–57) and i.p. injected to a tumor-bearing more efficient to use the highly basic region containing residues mouse, the biologically active fusion protein was specifically stabilized in 49–57. Through covalent attachment to TAT , several proteins solid tumors but was hardly detected in the normal tissue. Furthermore, 47–57 have been delivered into cells, including an inhibitor of human pap- when wild-type (WT) caspase-3 (Casp3WT) or its catalytically inactive illomavirus type 16 (19), ovalbumin into the MHC class I pathway mutant was fused to TAT-ODD and i.p. injected to a tumor-bearing kip1 INK4a mouse, the size of tumors was reduced by the administration of TAT- (20), the cyclin-dependent kinase inhibitors p27 (21) and p16 WT WT ODD-Casp3 but not by TAT-ODD-mutant Casp3. TAT-ODD-Casp3 (22), and a Casp3 protein (23). TAT79–57 has also been successfully did not cause any obvious side effects on tumor-bearing mice, suggesting used in vivo to deliver ␤-Gal into all tissues of the mouse, including specific stabilization and activation of the fusion protein in the hypoxic the brain (24). tumor cells. These results suggest that the combination of protein therapy Casp3 is a major executioner protease in known apoptotic path- using a cytotoxic TAT-ODD fusion protein with radiotherapy and chem- ways. Casp3 remains dormant until the initiator caspases activate it by otherapy may provide a new strategy for annihilating solid tumors. direct proteolysis. Activated Casp3 cleaves the inhibitor of caspase- activated DNase, resulting in cell death (25–27). Activation of Casp3 INTRODUCTION under hypoxic conditions has been reported in several cell types, such as ventricular myocytes, endothelial cells, and squamous cell lung Ͼ Solid tumors comprise 90% of all of human malignancies. Fur- carcinomas (28–31). thermore, biologically and therapeutically significant hypoxia occurs Here we demonstrated that part of the ODD domain conferred in many solid tumor masses. The hypoxic tumor cells are more oxygen-dependent stability to a fusion protein and that the covalent resistant to radiotherapy and chemotherapy than their well-oxygen- ␤ attachment of TAT47–57 and ODD548–603 to bioactive -Gal allowed ated counterparts and are capable of stimulating angiogenesis by the quick delivery and specific stabilization in a solid tumor. Furthermore, release of several growth factors (e.g., vascular endothelial growth the replacement of ␤-Gal with Casp3 reduced tumor masses without factor, angiogenin, and platelet-derived growth factor) for endothelial any obvious side effects, demonstrating the feasibility and efficacy of cells (1–3). Although the specific delivery of anticancer gene products protein therapy using a TAT-ODD fusion protein for solid tumors. to hypoxic regions of solid tumors has been studied extensively (4–9), an effective technique has not been established. 2 ␣ The transcription factor, HIF -1 , is an important mediator of the MATERIALS AND METHODS hypoxic response of tumor cells and controls the up-regulation of a number of factors vital for solid tumor expansion, including angio- Cell Cultures and Reagents. The human lung adenocarcinoma cell line genic factors, such as vascular endothelial growth factor (10). The A549, the human embryo kidney cell line HEK293, the human pancreatic ␣ cancer cell line CF/PAC-1, and the mouse fibroblast cell line NIH/3T3 were up-regulation of HIF-1 by decreased cellular O2 concentration in- volves increased mRNA expression, protein stabilization, nuclear obtained from American Type Culture Collection. A549, HEK293, and NIH/ localization, and trans-activation (11). Perhaps the most striking reg- 3T3 were maintained in 5% FCS-DMEM, and CF/PAC-1 was maintained in 10% FCS-Iscove’s modified Dulbecco’s medium (Life Technologies, Inc., ulation occurs exclusively at the level of protein expression via ␣ Rockville, MD) supplemented with penicillin (100 units/ml) and streptomycin changes in the half-life of the HIF-1 protein in response to hypoxia (100 ␮g/ml). Cbz-LLL (Peptide Institute, Inc., Osaka, Japan) was dissolved in both in cultured cells and in vivo (12–14). DMSO to 10 mM. Pimo hydrochloride (Hypoxyprobe-1; Natural Pharmacia International, Inc., Research Triangle Park, NC) was dissolved in normal saline Received 9/21/01; accepted 1/24/02. (10 mg/ml). The costs of publication of this article were defrayed in part by the payment of page DNA Constructs. To construct pCH/ODD plasmids encoding a series of charges. This article must therefore be hereby marked advertisement in accordance with ODD-␤-Gal fusion proteins, pCH was first constructed by annealing the 18 U.S.C. Section 1734 solely to indicate this fact. Ј 1 To whom requests for reprints should be addressed, at Department of Molecular oligonucleotides containing Kozak and NLS sequences (5 -AGCTTGACAT- Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo- GGCGCCTAAGAAGAAGAGGAAGA-3Ј and 5Ј-GATCTCTTCCTCTTCT- ku, Kyoto 606-8501, Japan. TCTTAGGCGCCATGTCA-3Ј) and inserting the annealed DNA fragment 2 The abbreviations used are: HIF, hypoxia-inducible factor; ODD, oxygen-dependent between HindIII and BglII sites of the pCH110 vector (Amersham Pharmacia degradation; ␤-Gal, ␤-galactosidase; NLS, nuclear localization signal; Cbz-LLL, N- carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal; Pimo, Pimonidazole; Ab, antibody; Biotech, Milwaukee, WI). Then cDNA fragments encoding various lengths of WT, wild type; Casp3, Caspase-3; MUT, mutant. the ODD domain of human HIF-1␣ were inserted between BglII and KpnI sites 2013 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. ANTITUMOR EFFECT OF TAT-ODD FUSION PROTEIN of pCH. The cDNA fragments encoding ODD were amplified by PCR using pCH plasmids or treated with TAT-fusion proteins were washed three times corresponding oligonucleotides as primers from cDNA prepared from total with PBS, fixed with 1% formaldehayde-0.2% glutaraldehyde solution, RNA from A549. The ODD fragments in pCH/557-574, pCH/562–569, pCH/ washed twice with PBS, and then treated with an X-Gal staining solution (5 557-571, and pCH/560-574 were prepared by annealing the corresponding mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mM MgCl2, and oligonucleotides. 0.04% X-gal) at 37°C. The experiments were repeated twice with similar To construct pBAD/PC, pBAD/3-0, and pBAD/557-574, which encode the results. ␤ -Gal protein fused to TAT, TAT-ODD/3-0, and TAT-ODD557–574, respec- Hypoxia. Hypoxia was achieved by incubating cells in a humidified environ- tively, pCH/TAT/PC, pCH/TAT/3-0, and pCH/TAT/557-574 were first con- ment at 37°CinCO2 incubators maintained at 94% N2,5%CO2, and 1% O2. structed. To prepare pCH/TAT/3-0, pCH/TAT/557-574, or pCH/TAT/PC, Preparation of TAT-fusion Protein and in Vitro Assay. To prepare ␤ oligonucleotides encoding TAT47–57 were inserted into the BglII site of pCH/ TAT-, TAT-ODD/3-0-, and TAT-ODD557–574- -Gal proteins, pBAD/PC, 3-0 and pCH/557-574 or substituted with the BglI-KpnI ODD/3-0 fragment of pBAD/3-0, and pBAD/557-574 were transformed into LMG194 bacteria (In- pCH/3-0, respectively. Then, pCH/TAT/3-0, pCH/TAT/557-574, and pCH/ vitrogen). TAT-fusion proteins were purified over a Ni2ϩ-NTA column (Qia- TAT/PC were digested with HindIII, blunted with klenow-fragment, and gen) following the manufacturer’s instruction manual. The ␤-Gal activities of further digested with SacI. The blunt-SacI DNA fragments were inserted
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