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[Cancer Research 65, 11622-11630, December 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

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

Nobuko Koshikawa1,2 and Keizo Takenaga1

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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{alpha} (HIF-1{alpha}) 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{alpha} 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-AW153F/ODD or DT-AH21A/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-AW153F/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-AW153F/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)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In most solid tumors, hypoxic areas are generated due to shortage of blood supply (1, 2). Because tumor cells in these hypoxic areas do not divide, they are resistant to conventional chemotherapy and radiotherapy (3). To make the matters worse, in vivo exposure of certain tumor cells to hypoxia followed by reoxygenation results in enhancement of invasive and metastatic potential (4). Recent studies have shown that tumor cells under hypoxic conditions overexpress Met and CRCX4 chemokine receptor and respond to hepatocyte growth factor and stromal cell–derived factor-1{alpha}, respectively, resulting in the stimulation of invasiveness and chemoattraction to organs that express stromal cell–derived factor-1{alpha} (5, 6). It has also been reported that p53–/– transformed cells and some of high-metastatic tumor cells are more resistant to hypoxia-induced apoptosis compared with p53+/+ cells and low-metastatic cells, respectively (7, 8). Mutations, genetic instability, DNA overreplication, and gene amplification are frequently induced in hypoxic cells (9–11). These data collectively indicate that hypoxic tumor cells exhibit more aggressive phenotype than normoxic ones and potentially become more malignant, and thus eradication of hypoxic tumor cells is inevitable to cure cancer patients.

Tumor cells in hypoxic microenvironments produce vascular endothelial growth factor (VEGF) to stimulate neoangiogenesis (12). Hepatoma cells also produce erythropoietin (Epo) in response to hypoxia (13). Hypoxic induction of VEGF and Epo is regulated at both transcriptional and posttranscriptional levels (14). Hypoxia activates the transcriptional complex termed hypoxia-inducible factor-1 (HIF-1), which is a heterodimer composed of an oxygen-regulated {alpha} subunit (HIF-1{alpha}) and a constitutively expressed ß-subunit (HIF-1ß/aryl hydrocarbon receptor nuclear translocator), both subunits being members of a subfamily of basic helix-loop-helix proteins that contain a conserved PAS domain (15, 16). HIF-1 binds to the hypoxia response element located upstream of the VEGF gene or downstream of the Epo gene and transactivates its expression (17). In normoxic conditions, HIF-1{alpha} is hydroxylated at two proline residues in the oxygen-dependent degradation (ODD) domain by HIF prolyl hydroxylases, recognized by the von Hippel-Lindau (pVHL)/Elongin B/Elongin C/Cul2 E3 ligase complex, and then subjected to rapid ubiquitination followed by proteasomal degradation (18, 19). When exposed to hypoxia, the activity of HIF prolyl hydroxylases is down-regulated and subsequently HIF-1{alpha} is rapidly stabilized (20, 21). The posttranscriptional regulation of VEGF and Epo mRNA involves the stabilization of the mRNA by the RNA-binding protein HuR and Epo mRNA-binding protein (ERBP), respectively, the binding site of which resides in the mRNA 3'-untranslated region (UTR; refs. 22, 23).

Hypoxia can be regarded as a physiologic abnormality that is restricted to the tumor; thus, it can be used as a trigger for heterologous gene expression. In fact, others and we have shown that hypoxia response element–regulated expression of prodrug-activating enzymes can sensitize hypoxic tumor cells to the corresponding prodrugs (24–27). Principally, however, this strategy is probably ineffective to quiescent tumor cells under hypoxic circumstances. Moreover, poor perfusion may limit prodrug diffusion to hypoxic regions. Therefore, to kill hypoxic tumor cells that are quiescent and insensitive to conventional chemotherapeutic agents, it is desirable to use a method that ensures cell death once a heterologous gene is expressed in the cells.

Diphtheria toxin A chain (DT-A) is the component of diphtheria toxin that inhibits protein synthesis in susceptible cells. It directly binds NAD+ and catalyzes the transfer of ADP ribose from NAD+ to elongation factor 2, which irreversibly inhibits elongation factor 2 (28). DT-A gene has been considered to be applicable for cancer gene therapy (29–34). Because DT-A leads to rapid cell cycle–independent death, it might be useful to kill hypoxic tumor cells if its expression is properly regulated. However, because of its extreme toxicity, it is hard to reduce nonspecific cytotoxicity (35). Nevertheless, several strategies that limit its toxicity have been developed and those include targeted delivery of DT-A to specific cells or tissue-specific expression, such as DT-A immunotoxin (30), DT-A fused to peptide ligands for cell-specific receptor (32), and DT-A expression construct under the control of a regulatory element or tissue-specific promoter (31, 33, 34). With regard to cancer gene therapy, {alpha}-fetoprotein promoter and prostate-specific antigen promoter–regulated expression of DT-A gene led to selective killing of hepatocarcinoma cell lines and prostate cancer cell lines, respectively, by using a liposomal gene transfer system (33, 34). In other cases, however, although preferential killing of target tumor cells could be shown, nonspecific cytotoxicity could not be abolished due to the background expression of DT-A (35).

In the present study, we examined the feasibility of application of DT-A gene for killing hypoxic tumor cells. To induce hypoxia-dependent expression of DT-A, we constructed an expression vector harboring wild-type or attenuated DT-A gene under the control of VEGF promoter. To increase hypoxia specificity, we designed the expression vector to express DT-A fused with HIF-1{alpha} ODD domain, expecting that the fusion protein is rapidly ubiquitinated and degraded through proteasome pathway in normoxia but stabilized in hypoxia. In addition, we constructed it to express mRNA containing ERBP-binding sequence (EBBS), aiming for stabilization of the mRNA in hypoxia. We report here that expression of attenuated DT-AW153F/HIF-1{alpha} ODD fusion protein driven by VEGF promoter causes efficient cell death of hypoxic tumor cells in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Cell Culture
Highly metastatic A11 cells and low-metastatic P29 cells are derived from Lewis lung carcinoma and their characteristics are described elsewhere (25). pVHL-deficient human renal carcinoma 786-O and their transfectants stably expressing wild-type pVHL (referred herein as 786-O/VHL cells) were generously provided by Dr. Y. Nagashima (Yokohama City University School of Medicine, Yokohama, Japan). Human hepatoma HepG2 cells were obtained from Human Science Research Resources Bank (Osaka, Japan). The cells were cultured at 37°C in DMEM supplemented with heat-inactivated 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. They were also cultured under hypoxic environment (1% O2) generated in NAPCO automatic O2/CO2 incubator (Precision Scientific, Chicago, IL). A11H10 cells were established after exposing A11 cells 10 times to severe hypoxia (<0.1% O2, generated in GasPak Pouch, Becton Dickinson, Cockeysville, MD) reoxygenation cycle.

Plasmid Constructions
pVEGFpro-DT-A/ODD-EBBS expression plasmid. To avoid the effect of enhancer/promoter sequence that an expression vector usually contains for driving a drug selection marker gene, we used the backbone of pGL3-basic (Promega, Madison, MD) to construct the DT-A/ODD expression vector. First, EBBS corresponding to 117 nucleotides downstream of the stop codon of Epo mRNA was prepared by reverse transcription-PCR (RT-PCR) using total RNA isolated from HepG2 cells and the sense primer 5'-CCAGGTGTGTCCACCTGGGC-3' and the antisense primer 5'-GACAGGCTGGCGCTGAGCTG-3'. The resulting PCR product was subcloned into pGEM-T Easy vector (Promega) and the insert was cut out with NotI, gel-purified, and then inserted into the NotI site of pcDNA3 to make a plasmid pcDNA3-ERBP. Next, the nucleotide sequence of ODD domain corresponding to the amino acid residues 396 to 618 of human HIF-1{alpha} was amplified by RT-PCR using total RNA isolated from HepG2 cells and the sense primer 5'-ACAGGGGACAGATGACCAGG-3' and the antisense primer 5'-TCAGGCGTCTTCCCAGCATG-3'. After subcloning in pGEM-T Easy vector, the insert was cut out with SalI and XhoI, gel-purified, and ligated to the SalI/XhoI cut pIBI-DT-A plasmid (kindly provided by Dr. Gail Harrison, University of Colorado Health Sciences Center, Denver, CO). The intrinsic stop codon TAG of DT-A gene was then changed to TTG by using Mutan-Express Km kit (TaKaRa Biomedicals, Osaka, Japan) and the oligonucleotide 5'-GGTCGACTCAAGAGGATCCC-3' so that DT-A fuses with ODD in frame. The resulting plasmid was digested with NruI and AatI, blunt-ended by T4 DNA polymerase, and ligated to the EcoRV-cut pcDNA3-EBBS to make a plasmid pcDNA3-DT-A/ODD-EBBS. The resulting plasmid was digested with NcoI and XbaI, and the DT-A/ODD-EBBS cassette was gel-purified, blunt-ended, and then ligated to the NcoI/XbaI cut, blunt-ended pGL3-basic to make a plasmid pGL3-DT-A/ODD-EBBS. The VEGF promoter sequence of the phVEGF-1 plasmid (kindly provided by Dr. H. Esumi, National Cancer Center Research Institute East, Tokyo, Japan; ref. 36) was digested with KpnI and NheI, gel-purified, and ligated to the KpnI/NheI-cut pGL3-basic or pGL3-DT-A/ODD-EBBS, yielding a plasmid pVEGFpro or pVEGFpro-DT-A/ODD-EBBS, respectively. The plasmid-harboring attenuated DT-A, pVEGFpro-DT-AW153F/ODD-EBBS, was made by changing the codon TGG to TTT, resulting in the amino acid change from tryptophan 153 to phenylalanine by using Transformer Site-Directed Mutagenesis kit (BD Sciences Clontech, Tokyo, Japan). pVEGFpro-DT-AH21A/ODD-EBBS was made in a similar way by changing the codon CAC to GCC, resulting in the amino acid change from histidine 21 to alanine. All of the nucleotide sequence prepared by RT-PCR and mutagenesis were confirmed by nucleotide sequencing.

Luciferase reporter plasmid harboring erythropoietin mRNA-binding protein-binding sequence. The EBBS in pGEM-T Easy prepared as above was digested with EcoRI, blunt-ended, and then ligated to the pflM1 cut, blunt-ended pGL2-basic (Promega). The VEGF promoter sequence was then inserted into the KpnI/NheI site of the plasmid to make a plasmid pVEGFpro-Luc-EBBS.

In vitro Ubiquitination Assay
In vitro ubiquitination assay was done as described previously (37). Briefly, DT-A and HIF-1{alpha} radiolabeled with 35SPRO-MIX (Amersham Biosciences Corp., Piscataway, NJ) were prepared by in vitro transcription and translation of genes subcloned into pcDNA3 using the TNT T7 Quick Coupled Transcription/Translation System (Promega). To prepare cell extracts (S100 fraction), 293T cells were washed twice with ice-cold hypotonic extraction buffer [20 mmol/L Tris-HCl (pH 7.5), 5 mmol/L KCl, 1.5 mmol/L MgCl2, and 1 mmol/L DTT]. After removal of the buffer, cells were disrupted in a Dounce homogenizer. Following cell lysis, crude extract was centrifuged at 10,000 x g for 10 minutes at 4°C. The supernatant was collected and further centrifuged at 100,000 x g for 4 hours at 4°C. The resulting cell extract (S100 fraction) was stored in aliquot at –80°C. Ubiquitination assays were done at 30°C in a total volume of 20 µL composed of 2.5 µL of programmed reticulocyte lysate, 83 µg of S100 fraction, 2 µL of 10 x ATP-regenerating system [20 mmol/L Tris-HCl (pH 7.5), 10 mmol/L ATP, 10 mmol/L magnesium acetate, 300 mmol/L creatine phosphate, and 0.5 mg/mL creatine phosphokinase], 1 µL of 10 mg/mL ubiquitin (Sigma-Aldrich, St. Louis, MO), or 10 mg/mL ubiquitin aldehyde (Boston Biochem, Cambridge, MA). Aliquots were removed at indicated times, mixed with SDS sample buffer, and analyzed by 12.5% SDS-PAGE followed by autoradiography.

In vitro Assay of Translation Inhibitory Activity of Diphtheria Toxin A
Translation inhibitory activities of DT-A, DT-A/ODD, and its attenuated fusion proteins were assayed as described (38) with some modifications. First, DT-A proteins were prepared by in vitro transcription/translation of genes subcloned into pcDNA3 using TNT T7 Quick Coupled Transcription/Translation System. Then, 1 µL of the translated DT-A protein was added to 11 µL of TNT SP6 Quick Coupled Transcription/Translation mixture containing 0.5 µg of NAD+, 0.45 µL 35SPRO-MIX, and 250 ng of luciferase SP6 control DNA (Promega). The samples were incubated for 90 minutes at 30°C and the proteins were separated on 12.5% SDS-PAGE and detected by autoradiography.

Luciferase Reporter Assay
For examining the effect of EBBS, the luciferase reporter plasmid, pVEGFpro-Luc or pVEGFpro-Luc-EBBS, was transiently transfected into cells using Lipofectin (Invitrogen Corp., Carlsbad, CA). As a control for transfection efficiency, pRL-CMV vector (Promega) was cotransfected with test plasmids. pGL2-control vector (Promega) was used as a positive control. Luciferase activity in cell extracts was assayed 48 hours posttransfection according to Dual-Luciferase reporter assay system protocols (Promega) using a luminometer (model TD-20/20, Turner Designs, Sunnyvale, CA).

RNA Extraction and Northern Blot Analysis
Total RNA was extracted with guanidinium thiocyanate from cells cultured under normoxic or hypoxic environment. Total RNA (20 µg) was electrophoresed on 1% agarose gel containing formaldehyde and transferred to nylon filters. Blots were hybridized with a 32P-labeled mouse VEGF cDNA probe, which was prepared by the random primer method. Filters were finally washed at 50°C in 30 mmol/L NaCl, 3 mmol/L sodium citrate, and 0.1% SDS.

Assays for Apoptosis
Chromatin condensation and fragmentation were visualized by staining the cells with 4',6-diamidino-2-phenylindole (DAPI, 10 µg/mL; ref. 8). Annexin V staining was done using Annexin V–enhanced green fluorescent protein (EGFP) apoptosis detection kit (MBL, Nagoya, Japan), according to the instructions of the manufacturer. Fluorescence was observed under a Fluoview confocal laser microscope (Olympus, Tokyo, Japan). Flow cytometric analysis was done as described previously (8) to analyze cellular DNA fragmentation with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Cell death was monitored by trypan blue dye exclusion.

In vivo Gene Transfer and Detection of Hypoxic Areas and Apoptosis in Tumors
A11H10 cells (5 x 105 cells) were inoculated s.c. into the abdominal flank of female C57BL/6 mice (Nippon SLC, Shizuoka, Japan). Twelve days after the inoculation, when an estimated tumor volume reached ~800 mm3, DNA/liposome complex that is composed of 40 µg of pEGFP-N1, pVEGFpro or pVEGFpro-DT-AW153F/ODD-ERBP, 40 µL of DMRIE-C (Life Technologies, Tokyo, Japan), and 5 µg of MW 70,000, lysine-fixable dextran-tetramethylrhodamine conjugates (dextran-TMR, Molecular Probes, Inc., Eugene, OR) in 200 µL Opti-MEM was directly injected intratumorally. Two days after the injection, 300 µL of EF5 solution (3 mg/mL) were administered i.p. into mice bearing s.c. tumors (39). One hour later, tumors were surgically removed and frozen in optimum cutting temperature compound. Cryostat sections cut at 10 µm were fixed with 4% paraformaldehyde and washed with Dulbecco's PBS (DPBS). To detect apoptotic tumor cells, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) stainings were done on the sections using ApopTag Fluorescein In situ Apoptosis Detection kit (Serologicals Corp., Norcross, GA), according to the instructions of the manufacturer. The sections were then treated with 5% mouse serum, 20% dry milk, and 0.3% Tween 20 in DPBS overnight at 4°C to block nonspecific binding sites. They were rinsed with 0.3% Tween 20 in DPBS and then incubated with Cy5-labeled monoclonal anti-EF5 antibody (ELK3-51) for 4 hours at 4°C to detect hypoxic regions in the tumors. After extensive washing with DPBS, tissue samples were counterstained with Hoechst 33324 and observed under a fluorescence microscope. Images were captured using a Cool SNAP charge-coupled device camera and processed by a RS IMAGE Express image processing software (Nippon Roper, Chiba, Japan).

In vivo Gene Transfer and Tumor Growth
P29 cells (4 x 105) were inoculated s.c. into the abdominal flank of female C57BL/6 mice (10 mice per group). Ten days after the inoculation, DNA/liposome complex (12 µg of pVEGFpro or pVEGFpro-DT-AW153F/ODD-ERBP and 3 µL of DMRIE-C in 200 µL of Opti-MEM) was directly injected into the tumor. The injection was done daily until the end of the experiments. Tumor growth was monitored by caliper measurement of two diameters at right angles and the tumor mass was estimated from the equation, volume = 0.5 x a x b2, where a and b are the larger and smaller diameters, respectively.

Determination of the Degree of Hypoxia in Subcutaneous Tumors
A11H10 and P29 cells (1 x 106) were inoculated s.c. into C57BL/6 mice. When tumor volumes reached ~650 mm3, the mice were injected with EF5. Cryostat sections of the tumors were made at every 400 µm distance and stained with Cy3-labeled monoclonal anti-EF5 antibody (ELK3-51) as described above. For analyzing EF5-positive (hypoxic) areas on each section, at least five randomly selected fluorescent images were captured with a Leica fluorescence microscope system equipped with a computer. The images were transferred to the ImageJ 1.34s software and EF5-positive areas were analyzed. In this way, >100 fields (1.175 mm2/field) were analyzed and the percentage of EF5-positive area per field was calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oxygen and von Hippel-Lindau–dependent ubiquitination of DT-A/ODD fusion protein. To examine the effect of HIF-1{alpha} ODD domain on ubiquitination of DT-A/ODD fusion protein, we subjected the in vitro translated 35S-labeled DT-A/ODD proteins to the in vitro ubiquitination assay. Incubation of DT-A/ODD with cell extract from 293T cells alone resulted in the appearance of a slower migrating form (Fig. 1A). The effect was enhanced by the addition of ATP generation system. Addition of ubiquitin resulted in a further mobility shift of these species. These results indicated that these mobility shifts were most likely due to ubiquitination of DT-A/ODD proteins. This was confirmed by a further shift of these species to high molecular weight proteins by the addition of ubiquitin aldehyde, an isopeptidase inhibitor that prevents the degradation of ubiquitin conjugates. Ubiquitination of DT-A/ODD, but not of DT-A that served as a control, proceeded as the incubation time prolonged (Fig. 1B), which was comparable with that of HIF-1{alpha} proteins (Fig. 1C). Addition of desferrioxamine, a hypoxia mimetic, to the reaction mixture inhibited DT-A/ODD ubiquitination in a dose-dependent manner (Fig. 1D). Furthermore, ubiquitination of the protein was more prominent in 786-O/VHL cells than in 786-O cells, although a low level of DT-A/ODD ubiquitination was apparent in 786-O cells (Fig. 1E). Taken together, these results indicate that DT-A/ODD, but not DT-A, is ubiquitinated dependently on oxygen and pVHL.



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  		Figure 1. Oxygen- and pVHL-dependent ubiquitination of DT-A/ODD. A, in vitro ubiquitination of DT-A/ODD. 35S-labeled DT-A/ODD was subjected to in vitro ubiquitination in reactions of different composition. Additions are indicated as follows: 293T S100 fraction (cell extract), ATP-regenerating system (ATP), ubiquitin (Ub), and ubiquitin aldehyde (UbAl). The mixture was incubated for 4 hours at 30°C. B, time course of ubiquitination of DT-A and DT-A/ODD proteins. 35S-labeled DT-A and DT-A/ODD proteins were subjected to in vitro ubiquitination in reactions containing all components described above. C, ubiquitination of HIF-1{alpha}. 35S-labeled HIF-1{alpha} was subjected to in vitro ubiquitination assay as in (B) for the indicated period. D, oxygen-dependent ubiquitination of DT-A/ODD. 35S-labeled DT-A/ODD was subjected to in vitro ubiquitination assay in the presence or absence of ubiquitin. The mixture was incubated for 4 hours in the presence of the indicated concentrations of desferrioxamine (DFO). E, pVHL-dependent ubiquitination of DT-A/ODD. 35S-labeled DT-A/ODD was subjected to in vitro ubiquitination assay as in (B) for the indicated period, except using S100 fraction prepared from 786-O or 786-O/VHL cells.

 
Effect of erythropoietin mRNA–binding protein binding sequence on the expression level of luciferase under hypoxic conditions. To examine the effect of EBBS on the expression level of a heterologous gene, we constructed luciferase reporter plasmids, pVEGFpro-Luc as a control and pVEGFpro-Luc-EBBS (Fig. 2A). After transient transfection of each reporter plasmid into A11 or HepG2 cells, they were exposed to hypoxia for 18 hours and then luciferase activity was measured. As shown in Fig. 2B and C, although integration of EBBS into the reporter plasmid resulted in an increase in the luciferase activity under normoxic conditions, it greatly enhanced the activity under hypoxic conditions in both cells. Similar results were obtained in human mammary carcinoma MCF7 cells and human foreskin fibroblasts (data not shown). Thus, EBBS caused a marked enhancement in luciferase activity in different cell types in hypoxia probably due to its mRNA-stabilizing effect.



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  		Figure 2. Effect of EBBS on the expression level of a heterologous gene under hypoxic conditions. A, schematic representation of the luciferase reporter constructs. VEGFpro, VEGF gene promoter. B and C, effect of EBBS on the expression level of a luciferase reporter gene. A11 or HepG2 cells cotransfected with pVEGFpro-Luc or pVEGFpro-Luc-EBBS and pRL-CMV as an internal control were cultured for 18 hours under normoxic (N) or hypoxic (H) conditions. All luciferase activities were normalized for transfection efficiency and background luciferase activities obtained from cells transfected with a promoterless luciferase gene pGL2-basic were subtracted from this value. Columns, luciferase activity expressed in arbitrary units; bars, SD of triplicate determinations.

 
In vitro protein synthesis inhibition by diphtheria toxin A, DT-A/ODD, and its attenuated fusion proteins. We next examined whether fusion of HIF-1{alpha} ODD domain to DT-A affects the translation inhibitory activity of DT-A. For this, we first made in vitro translated DT-A and DT-A/ODD and then added them to the in vitro luciferase gene transcription/translation system with or without exogenously added NAD+. Figure 3 shows the amount of translated 35S-labeled luciferase protein. DT-A significantly reduced the level of luciferase protein even in the absence of exogenous NAD+ and nearly completely inhibited the translation in the presence of NAD+. DT-A/ODD was comparable with DT-A in inhibiting translation. We also made attenuated DT-A/ODD proteins, DT-AW153F/ODD and DT-AH21A/ODD, and measured their translation inhibitory activities in a similar way. DT-AW153F and DT-AH21A have been reported to have about a 4-fold and 120-fold reduced ADP ribosyltransferase activity compared with DT-A, respectively (40, 41). Consistent with the reports, DT-AW153F/ODD was less effective in inhibiting the synthesis of luciferase protein compared with DT-A/ODD. DT-AH21A/ODD exhibited the least inhibitory activity.



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  		Figure 3. In vitro protein synthesis inhibitory activity of wild-type and attenuated DT-A/ODD fusion protein. DT-A/ODD, DT-AW153F/ODD, or DT-AH21A/ODD prepared by TNT T7 Quick Coupled Transcription/Translation System was added to TNT SP6 Quick Coupled Transcription/Translation mixture composed of [35S]PRO-MIX and luciferase SP6 control DNA. The reaction mixtures were incubated for 90 minutes at 30°C in the presence or absence of NAD+. Newly synthesized luciferase protein was separated on SDS-PAGE and detected by autoradiography.

 
Induction of apoptosis by DT-A/ODD proteins in A11 cells under hypoxic conditions. Based on the above results, we constructed DT-A expression plasmids, pVEGFpro-DT-A/ODD-EBBS, pVEGFpro-DT-AW153F/ODD-EBBS, and pVEGFpro-DT-AH21A/ODD-EBBS. To investigate the effect of these expression plasmids on apoptosis of A11 cells under normoxic and hypoxic conditions, we transfected each of them into A11 cells, cultured the cells under normoxic or hypoxic conditions for 24 hours, and then stained them for Annexin V. The transfection efficiency was 20% to 30% as assessed by pEGFP-N1 expression vector. As shown in Fig. 4A, hypoxia alone did not induce apoptosis in the control plasmid-transfected cells, thus excluding nonspecific induction of apoptosis. The reason why ~10% of Annexin V–positive cells were observed in the control plasmid-transfected cells is unknown; however, it may be due to the toxicity of the transfection reagent. Expression of DT-A/ODD proteins resulted in a significant increase in the number of Annexin V–positive cells irrespective of oxygen tension. Expression of DT-AW153F/ODD or DT-AH21A/ODD did not apparently induce apoptosis under normoxic conditions. In contrast, they significantly caused an increase in the number of Annexin V–positive cells under hypoxic conditions, DT-AW153F/ODD being more potent than DT-AH21A/ODD (Fig. 4A and B). Similar results were obtained in their abilities to induce sub-G1 fraction, another criteria of apoptosis, under hypoxic conditions (Fig. 4C). Long-term exposure of A11 cells to severe hypoxia resulted in cell death to some extent, but expression of DT-AW153F/ODD further enhanced cell death (Fig. 4D). Thus, transfection of pVEGFpro-DT-AW153F/ODD-EBBS or pVEGFpro-DT-AH21A/ODD-EBBS plasmid into A11 cells led to a clear hypoxia-dependent induction of apoptosis.



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  		Figure 4. Induction of apoptosis in A11 cells transfected with pVEGFpro-DT-A/ODD-EBBS, pVEGFpro-DT-AW153F/ODD-EBBS, and pVEGFpro-DT-AH21A/ODD-EBBS vectors. A11 cells were transiently transfected with pVEGFpro as a control, pVEGFpro-DT-A/ODD-EBBS, pVEGFpro-DT-AW153F/ODD-EBBS, and pVEGFpro-DT-AH21A/ODD-EBBS. After culturing the cells under normoxic or hypoxic conditions for 24, 36, or 48 hours, the cells were processed for Annexin V staining (A and B), sub-G1 analysis (C), or trypan blue dye exclusion test (D), respectively. A, induction of Annexin V–positive cells by the expression vectors. B, Annexin V–positive cells induced by the transfection of pVEGFpro-DT-AW153F/ODD-EBBS in normoxia (left) or hypoxia (right). The fluorescence was observed under a confocal laser microscope. C, induction of cellular DNA fragmentation (sub-G1) by the expression vectors in hypoxia. Columns, percentage of sub-G1 fraction determined with a FACScan flow cytometer. D, induction of cell death by the expression vectors in severe hypoxia. Columns, percentage of dead cells; bars, SD of triplicate determinations. *, P < 0.007.

 
Establishment of A11H10 cells with higher resistance to hypoxia-induced apoptosis. We next wanted to investigate the effect of liposomal gene transfer of the DT-A/ODD expression construct on induction of apoptosis in hypoxic regions of tumors. Before this experiment, we established A11H10 cells with higher resistance to hypoxia-induced apoptosis than A11 cells and used them as target cells. The cells were actually more resistant to apoptosis induced by severe hypoxia (<0.1% O2) than A11 cells (Fig. 5A) and survived for >5 days in severe hypoxia (Fig. 5B). They were also more resistant to apoptosis triggered by an anticancer drug cisplatin than A11 cells (Fig. 5C). The apoptosis-resistant phenotype was stable at least for 3 months (data not shown). A11H10 cells expressed VEGF mRNA as well as A11 cells in response to hypoxia (Fig. 5D).



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  		Figure 5. Establishment and characterization of A11H10 cells. A, apoptosis of A11 and A11H10 cells in severe hypoxia. The cells were cultured in normoxia or severe hypoxia (<0.1% O2; H) for 28 hours and the percentage of cells with chromatin condensation and fragmentation was determined after DAPI staining. Columns, percentage of apoptotic cells; bars, SD of triplicate determinations. B, survival of A11 and A11H10 cells in severe hypoxia. The cells (1 x 105) were cultured in severe hypoxia (<0.1% O2) for 5 days followed by culturing in normoxia for 7 days. Colonies were stained with crystal violet. C, sensitivity of A11 and A11H10 cells to cisplatin. The cells were exposed to the indicated concentrations of cisplatin for 2 days. Cell viability was assessed by trypan blue dye exclusion. Points, percentage of viable cells; bars, SD of triplicate determinations. D, VEGF mRNA expression in A11 and A11H10 cells. Total RNA was isolated from the cells cultured in normoxia or hypoxia for 8 hours and subjected to Northern analysis. Blots were hybridized with 32P-labeled mouse VEGF cDNA. Ethidium bromide staining of the gel is also shown.

 
Apoptosis induction by liposomal transfer of diphtheria toxin A expression plasmid in hypoxic regions of A11H10 tumors. From the results described above, we chose the pVEGFpro-DT-AW153F/ODD-EBBS expression construct and examined its effect on apoptosis induction in tumors. Before this experiment, we directly injected pEGFP-N1/DIMRIE-C complex in A11H10 tumors along with lysine-fixable dextran-TMR to see if we can use dextran-TMR for later identification of injected areas. Two days after the injection, tumors were surgically removed and cryosections were prepared. Observation of the specimen revealed that EGFP-expressing tumor cells located in dextran-TMR–positive areas, indicating that dextran-TMR can be used to identify injected areas (Fig. 6). We then intratumorally injected pVEGFpro or pVEGFpro-DT-AW153F/ODD-EBBS/DMRIE-C complex together with dextran-TMR, and 2 days after the injection we administered EF5 for discrimination between normoxic and hypoxic regions. Cryosections were prepared and processed for TUNEL staining followed by staining with Cy5-labeled anti-EF5 antibody and Hoechst staining. We omitted necrotic areas from the analyses. The results showed that a few TUNEL-positive cells were observed in dextran-TMR–negative areas of both control and pVEGFpro-DT-AW153F/ODD-EBBS plasmid-injected tumors (Fig. 7A and C), whereas a large number of TUNEL-positive cells were detected in dextran-TMR-positive areas of the pVEGFpro-DT-AW153F/ODD-EBBS-injected tumors (Fig. 7D) but not of the pVEGFpro-injected tumors (Fig. 7B). Closer observations of the specimen of the pVEGFpro-DT-AW153F/ODD-EBBS-injected tumors revealed that only a small number of TUNEL-positive cells were evident in dextran-TMR–positive and EF5-negative (normoxic) areas (Fig. 8A-D), whereas a large number of TUNEL-positive cells were detected in dextran-TMR-positive and EF5-positive (hypoxic) areas (Fig. 8E-H). Thus, these results indicate that apoptosis was remarkably induced by liposomal transfer of pVEGF-DT-AW153F/ODD-EBBS expression construct in hypoxic, but not in normoxic, regions of A11H10 tumors.



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  		Figure 6. Liposomal gene transfer of pEGFP-N1 into A11H10 tumors. pEGFP-N1/DMRIE-C complex was directly injected into s.c. A11H10 tumors along with dextran-TMR. Two days after the injection, cryosections were prepared, stained with Hoechst 33342, and then observed under a fluorescent microscope for EGFP (green), dextran-TMR (orange), and Hoechst 33342 (blue). Images were captured using a Cool SNAP charge-coupled device camera and pseudocolored by a RS IMAGE Express image processing software. A, EGFP. B, dextran-TMR. C, Hoechst33342. D, merged. Note that EGFP-expressing cells locate in dextran-TMR-positive areas. Bar, 50 µm.

 


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  		Figure 7. Apoptosis induction by liposomal gene transfer of pVEGFpro-DT-AW153F/ODD-EBBS into A11H10 tumors. pVEGFpro as a control (A and B) or pVEGFpro-DT-AW153F/ODD-EBBS/DMRIE-C complex (C and D) was directly injected into A11H10 tumors along with dextran-TMR. Two days after the injection, cryosections were prepared, stained for TUNEL, and observed under a confocal microscope for TUNEL-positive cells (green) and dextran-TMR (orange). A and C, dextran-TMR–positive areas. B and D, dextran-TMR–negative areas. Bar, 100 µm.

 


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  		Figure 8. Apoptosis induction in hypoxic areas of A11H10 tumors after liposomal gene transfer of pVEGFpro-DT-AW153F/ODD-EBBS. pVEGFpro-DT-AW153F/ODD-EBBS/DMRIE-C complex was directly injected into A11H10 tumors along with dextran-TMR. Two days after the injection, the mice were administered with EF5. The cryosections of tumors were successively processed for TUNEL, EF5, and Hoechst 33342 stainings. Images were captured using a Cool SNAP charge-coupled device camera and pseudocolored by a RS IMAGE Express image processing software. TUNEL (green), EF5 (pink), Hoechst 33342 (blue), and dextran-TMR (orange). A to D, dextran-TMR–positive and EF5-negative areas. E to H, dextran-TMR–positive and EF5-positive areas. A and E, dextran-TMR. B and F, EF5. C and G, merged image of TUNEL and Hoechst 33342. D and H, merged image of TUNEL, EF5, and dextran-TMR. Bar, 50 µm.

 
Tumor growth inhibition by liposomal transfer of diphtheria toxin A expression plasmid in P29 tumors. Based on the above results, we repeatedly injected pVEGF-DT-AW153F/ODD-EBBS/liposome complex into A11H10 tumors to see the therapeutic effect of the plasmid. However, the inhibition of tumor growth was marginal (data not shown). We speculated that this is due to the fact that A11 tumors are well vascularized (42) and, hence, not so hypoxic. We then used P29 tumors because they are poorly vascularized (42). Actually, as expected, EF5 staining showed that P29 tumors contained increased hypoxic areas compared with A11H10 tumors (Fig. 9A and B). We therefore examined the effect of multiple injections of the expression construct on P29 tumor growth. The results showed that a modest but statistically significant growth inhibition was observed in the mice with pVEGF-DT-AW153F/ODD-EBBS compared with the mice with pVEGFpro without apparent side effects, such as body weight loss (Fig. 10A and B).



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  		Figure 9. The degree of intratumoral hypoxia in A11H10 and P29 tumors. A, detection of hypoxic areas in tumors by EF5 staining. Frozen sections of s.c. tumor established from A11H10 and P29 cells were stained for EF5. Bar, 100 µm. B, the degree of hypoxia in A11H10 and P29 tumors. Frozen sections prepared from different regions of A11H10 and P29 tumor were stained for EF5. The fluorescent images of >100 fields (1.175 mm2/field) were analyzed and the percentage of EF5-positive area per field (columns) was calculated. Bars, SE. *, P < 0.0001.

 


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  		Figure 10. Growth inhibition of P29 tumors by liposomal gene transfer of pVEGFpro-DT-AW153F/ODD-EBBS. A, growth of tumors injected with pVEGFpro ({circ}) or pVEGFpro-DT-AW153F/ODD-EBBS/DMRIE-C complex ({bullet}). The liposome complex was directly injected into the tumor daily. B, body weight of the mice with pVEGFpro ({circ}) or pVEGFpro-DT-AW153F/ODD-EBBS ({bullet}). Bars, SE. *, P < 0.04.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we examined the effectiveness of VEGF promoter–driven DT-A expression for killing hypoxic tumor cells. To reduce cytotoxicity of DT-A in normal cells and increase hypoxia specificity, we constructed the expression vector to express DT-A/ODD fusion protein to produce mRNA containing the EBBS and not to include unnecessary enhancer/promoter sequence other than VEGF promoter. The results showed that DT-A/ODD, but not DT-A, was ubiquitinated as well as HIF-1{alpha} in a pVHL- and oxygen-dependent manner in in vitro ubiquitination assays. The EBBS exhibited mRNA-stabilizing effect, as shown by luciferase reporter assays. Thus, HIF-1{alpha} ODD domain and the EBBS worked accordingly. In addition, DT-A/ODD exhibited a comparable inhibitory activity of protein synthesis with DT-A in vitro, indicating that fusion of the ODD domain to DT-A does not affect its translation inhibitory activity.

The ODD domain of HIF-1{alpha} has been implicated to be useful as a hypoxia switch, limiting the production of a fusion protein in normoxia while stabilizing it in hypoxia (43). For example, ODD–caspase-3 fusion protein is shown to be specifically stabilized and activated in hypoxic cells (44). The present study also showed that the ODD domain is capable of regulating the toxicity of attenuated DT-A, probably rendering the fusion protein to be recognized by pVHL and subsequently degraded through proteasome pathway. However, we presently have no evidence that these events actually occur in the cells.

ERBP was initially identified as the protein(s) that specifically binds to a 120 base fragment (EBBS) of the 3' UTR of Epo mRNA. ERBP binds to this sequence and prolongs the half-life of Epo mRNA as well as other reporter mRNA containing EBBS in normoxia and further in hypoxia (23). Although Epo is mainly expressed in cells derived from the liver and the kidney, ERBP is present in other tissues, such as lung, brain, and spleen (23). We could also observe the reporter mRNA-stabilizing effect of EBBS in lung carcinoma cells, hepatoma cells, breast carcinoma cells, and fibroblasts. Therefore, EBBS is able to stabilize a heterologous transcript in a variety of cell types. An alternative to stabilize mRNA in hypoxia may be the use of HuR-binding sequence or recently identified PAIP2-binding sequence, both of which are present in VEGF mRNA (22, 45). These elements could substitute for the EBBS in the present expression vector.

Transfection of pVEGFpro-DT-A/ODD-EBBS expression vector into high-metastatic A11 cells resulted in a marked induction of apoptosis in both normoxia and hypoxia. This indicated that only a background expression level of DT-A/ODD is too cytotoxic to be regulated. We then sought to use attenuated DT-A/ODD, DT-AW153F/ODD, and DT-AH21A/ODD, expecting that although they are less active in inhibiting protein synthesis than DT-A/ODD under normoxic conditions, they may exhibit a significantly higher activity when expressed and accumulated in the cells under hypoxic conditions. The results showed that DT-AW153F/ODD and DT-AH21A/ODD were very weak in inducing apoptosis under normoxic conditions, whereas they significantly induced apoptosis under hypoxic conditions. Thus, we could observe hypoxia-dependent induction of apoptosis in A11 cells transfected with the attenuated DT-A/ODD expression vector. The pVEGFpro-DT-AW153F/ODD-EBBS was superior to pVEGFpro-DT-AH21A/ODD-EBBS in inducing apoptosis.

Given that the pVEGFpro-DT-AW153F/ODD-EBBS vector strongly induce apoptosis in the transfected cells under hypoxic but not normoxic conditions, we decided to apply pVEGFpro-DT-AW153F/ODD-EBBS vector to in vivo experiments. Before investigating the effect, we established A11H10 cells that are highly resistant to hypoxia-induced apoptosis. We previously reported that A11 cells are more resistant to apoptosis induced by microenvironmental stresses than low-metastatic counterparts (8). A11H10 cells were further more resistant than A11 cells to not only hypoxia but also anticancer drugs and various ER stresses (glucose deprivation, tunicamycin, brefeldin A, calcium ionophore A23187; data not shown). Therefore, the cells enabled us to examine the effect of the expression vector on the tumors that are more difficult to cure. After establishing s.c. A11H10 tumors, we directly injected pVEGFpro-EBBS/liposome complex as a control or pVEGFpro-DT-AW153F/ODD-EBBS/liposome complex with dextran-TMR conjugates, which are frequently used for long-term tracers of live cells. Dextran-TMR conjugates were not toxic and could be detected in the tumors at least 3 days after the injection (data not shown) and, therefore, it was useful to monitor the injection areas of DNA/liposome complex. Actually, EGFP was expressed in cells located in dextran-TMR–positive areas in the tumors administrated with pEGFP-N1 plasmid/DMRIE-C complex. To detect hypoxic areas in the tumors, we used EF5, a derivative of nitroimidazole (39). The results showed that pVEGFpro-EBBS/liposome administration hardly induced apoptotic (TUNEL-positive) cells in dextran-TMR–positive areas. In contrast, pVEGFpro-DT-AW153F/ODD-EBBS/liposome administration resulted in a striking induction of apoptosis in dextran-TMR–positive areas. More specifically, apoptotic cells were detected in hypoxic, but not in normoxic, areas of pVEGFpro-DT-AW153F/ODD-EBBS/liposome-administered tumors. These results clearly indicate that intratumoral injection of pVEGFpro-DT-AW153F/ODD-EBBS/liposome complex induced apoptosis in a hypoxia-dependent fashion. It should be noted, however, that we could not observe a clear tumor growth inhibition after multiple injection of pVEGFpro-DT-AW153F/ODD-EBBS/liposome complex into a tumor mass. This contrasts to the case of direct injection of {alpha}-fetoprotein promoter/enhancer-driven DT-A expression construct/DMRIE-C complex into hepatoma in which significant growth retardation was observed (33). However, our results rather seem to reflect the hypoxia specificity of our expression construct. Because total hypoxic areas in an A11H10 tumor mass are small relative to total normoxic areas, an obvious growth inhibition may not be observed even if all hypoxic tumor cells are killed. Then, to test the therapeutic effect of the expression vector in more hypoxic tumors, we chose P29 tumors that have poorer vasculature and, thus, are more hypoxic than A11 tumors. As a result, we could clearly observe growth retardation of the tumor. Although the effect was not so striking, it may be due to the hypoxia specificity and inefficient in vivo gene transfer.

The present DT-AW153F/ODD expression vector would be applicable to a wide variety of tumors, especially those with severe hypoxia, such as cervical cancer and melanomas (46). These cancers are also suitable for intratumoral gene delivery. A critical point to be improved in the future is the transfection method of the vector into hypoxic tumor cells. Although liposomes used here have many advantages as a gene transfer method, such as low evocation of inflammations and immune responses, transfection efficiency is not so high. A solution to this point may be the use of adenoviruses and recently developed DT-resistant packaging line (34), which may allow us to produce a high titer of adenoviruses encoding DT-AW153F/ODD. Combination of such adenoviruses and conventional therapeutic regimens may lead to total killing of tumor cells and ultimately prevention of tumor recurrence.


   Acknowledgments
 
Grant support: Grant-in-aid from the Ministry of Health, Labour, and Welfare for Third Term Comprehensive Control Research for Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. S. Fujimoto for his support.

Received 1/13/05. Revised 8/24/05. Accepted 9/30/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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