Novel, chimeric, cancer-specific, and radiation-inducible gene promoters for suicide gene therapy of cancer

Authors

  • Jie Xiong MD, PhD,

    1. Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Wen-jie Sun MD, PhD,

    1. Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China
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    • The first 2 authors contributed equally to this article.

  • Wei-feng Wang MD,

    1. Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Zheng-kai Liao MD, PhD,

    1. Department of Radio-Chemotherapy, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Fu-xiang Zhou MD, PhD,

    1. Department of Radio-Chemotherapy, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Hai-yan Kong MD,

    1. Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Yu Xu MD, PhD,

    1. Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Cong-hua Xie MD, PhD,

    1. Department of Radio-Chemotherapy, Zhongnan Hospital, Wuhan University, Wuhan, China
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  • Yun-feng Zhou MD, PhD

    Corresponding author
    1. Department of Radio-Chemotherapy, Zhongnan Hospital, Wuhan University, Wuhan, China
    • Department of Radio-Chemotherapy, Zhongnan Hospital, Wuhan University, 169 Donghu Road, Wuhan 430071, China

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    • Fax: +86 27 6781 2889


Abstract

BACKGROUND:

Although the promoter of the human telomerase reverse transcriptase (hTERT) gene has been widely used in gene therapy for targeted cancer cells, it has some limitations for clinical use because of its low activity and potential toxicity to certain normal cells. To overcome these defects, the authors generated novel chimeric hTERT promoters that contained the radiation-inducible sequence CC(A/T)6GG (known as CArG elements).

METHODS:

Chimeric hTERT promoters were synthesized that contained different numbers of CArG elements, and the activity of chimeric promoters was assessed in different cancer cell lines and normal cells. The potential of selected promoters to successfully control horseradish peroxidase (HRP) and prodrug indole-3-acetic acid (IAA) suicide gene therapy was tested in vitro and in vivo.

RESULTS:

The promoter activity assays indicated that the synthetic promoter that contained 6 repeating CArG units had the best radiation inducibility than any other promoters that contained different numbers of CArG units, and the chimeric promoters retained their cancer-specific characteristics. The chimeric promoter was better at driving radiation-inducible gene therapy than the control promoters. The sensitizer enhancement ratio of the chimeric promoter system determined by clonogenic assay was higher, and the chimeric promoter system resulted in a significantly higher apoptotic level compared with other promoter systems. The combination of chimeric/promoter-mediated gene therapy and radiotherapy significantly inhibited tumor volume in a xenograft mouse model and resulted in a significant prolongation of survival in mice.

CONCLUSIONS:

The current results indicated that a combinational cancer-specific promoter system that is responsive to irradiation has great potential for improving the efficacy of cancer treatment. Cancer 2011;. © 2011 American Cancer Society.

Telomeres are special structures at the ends of eukaryotic chromosomes that appear to function in chromosome protection, positioning, and replication.1 Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA onto chromosomal ends using a segment of its RNA components as template.2 High activity of telomerase is detected in approximately 90% of all malignant tumors, making it a highly attractive target for the development of cancer-specific therapy.3 Human telomerase is composed of at least 2 components, a template RNA component (human telomerase RNA [hTR]) and a catalytic subunit, the human telomerase reverse transcriptase (hTERT). The expression of hTR is ubiquitous in all types of human cells regardless of the status of telomerase activity and acts as a template for telomere synthesis. hTERT is expressed only in cells and tissues that are positive for telomerase activity,4 strongly indicating that the targeting of hTERT represents a potential strategy for the treatment of tumor cells.

Recently, the promoter region of cancer-specific hTERT was cloned and characterized.5, 6 The promoter region of cancer-specific hTERT was GC rich and lacked both TATA and CAAT boxes. A major objective of cancer gene therapies is to target the expression of therapeutic genes in tumor cells selectively and specifically while sparing normal tissue from damage. Therefore, the feasibility of using the hTERT promoter to induce tumor-specific transgene expression in cancer gene therapy has been expected. Several studies have used hTERT promoter to drive the expression of therapeutic genes in different models of gene therapy,7, 8 but gene expression driven by the hTERT promoter is much lower than expression driven by commonly used viral promoters, such as cytomegalovirus (CMV) early promoter, and simian virus 40 (SV40) early promoter.9 Furthermore, expression of hTERT is observed at high levels in germ line cells and in cells with proliferative capacity, such as stem cells or epithelial cells of the mucosal lining in intestines,10, 11 and potential toxicity to these normal cells has been recognized as 1 of the major concerns regarding the use of the hTERT promoter to drive proapoptotic or cytotoxic gene expression. Thus, gene therapy strategies using a single hTERT promoter may have some limitations for clinical application and need further improvement.

Gene promoters activated by ionizing radiation (IR) can provide suitable control over the expression of therapeutic genes. Moreover, the cellular damage induced by IR can be exploited for the purposes of combinational gene therapy. The radiation-responsive promoter from the early growth response gene Egr1 has been used successfully for selective transgene expression after IR.12 The sequence CC(A/T)6GG, known as a CArG element, has been identified as a radiation-responsive motif within the Egr1 promoter, and different numbers of CArG elements may affect the activation response.13, 14 With a view to controlling the expression of therapeutic gene through irradiation and combining cancer-specific gene therapy with radiotherapy, we constructed and characterized novel chimeric hTERT promoters that contained different numbers of CArG elements.

In the current study, we explored a suicide gene therapy strategy, a gene-directed enzyme prodrug therapy (GDEPT) system consisting of horseradish peroxidase (HRP) and the prodrug indole-3-acetic acid (IAA), controlled by radiation-inducible and cancer-specific synthetic promoters. Delivery of the HRP gene to cancer cells followed by treatment with the prodrug IAA could provide a novel cancer GDEPT approach, and the HRP/IAA system results in fast and effective cancer cell kill in both toxic and hypoxic conditions and elicits a bystander effect.15, 16 The effect of chimeric promoter-controlled gene therapy combined with irradiation was detected in vitro and in vivo in this study.

MATERIALS AND METHODS

Cell Culture

Human cervical cancer cells (HeLa), human lung adenocarcinoma cells (A549), human hepatoma carcinoma cells (MHCC97), and normal human embryonic lung cells (hEL) were obtained from the China Center for Type Culture Collection. Cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco) and incubated in a humidified incubator at 37°C with 5% CO2.

Vector Construction

All restriction and modifying enzymes were supplied by either Promega (Madison, Wis) or TAKARA Bio USA (Madison, Wis) and were used according to the manufactures' instructions. DNA isolation and purification were carried out using appropriate kits from Omega Bio-Tek, Inc. (Norcross, Ga). The core hTERT promoter (phTERT) (−385/+40 base pairs) was kindly provided by Dr. I. Horikawa5 and was cloned into the pGL3-Basic vector (Promega) to yield the phTERT-luciferase (Luc) promoter. New promoters carrying 4, 6, 8, 10 tandem-repeat copies of a prototype CArG sequence (CCTTATTTGG) were constructed by using the complementary oligodeoxyribonucleotides pair (from Shanghai Shenggong Company, Shanghai, China). For example, the 5′-CGCGTGATAT(CCTTATTTGG)4-3′ and 5′-AATT(CCAAATAAGG)4ATATCA-3′ pair was cloned into the phTERT-luciferase by using MluI and EcoRI sites, and we denoted the vector that contained 4 CArG element enhancer repeat copies as pC4-hTERT-Luc. The same procedure was used to produce pC6-hTERT-Luc, pC8-hTERT-Luc, and pC10-hTERT-Luc. HRP combinational DNA (cDNA) excised from pRK5-HRP (kindly provided by Dr. Cutler17) by BamHI digestion was inserted into the multiple cloning site directly downstream of the promoter, and the vector pControl-HRP also was constructed as a positive control by cloning an HRP fragment into pControl-Luc (pGL3-control [Promega] containing SV40 promoter and enhancer) using the Hind III site.

Cell Transfection, Irradiation, and Luciferase Assay

HeLa, A549, MHCC97, and hEL cells were transiently transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. For each well in a 24-well plate, 1 × 105 cells were transfected with 0.4 μg of the promoter-reporter constructs, and 0.4 μg of the pRL-TK plasmid (Promega) was used as an internal control. Twenty-four hours after transfection, the cells were irradiated with 0 to 10 grays (Gy) of 60Co γ-rays (60Co therapeutic machine, GWXJ80 type; Nuclear Power Institute of China, China). After radiation treatment, the cells were reincubated, and luciferase activity was measured on a luminometer (GLOMA) 24 hours later, using the Dual-Luciferase Reporter Assay System (Promega).

Western Blot Analysis

Three types of cancer cells (HeLa, A549, and MHCC97) and 1 type of normal cells (hEL), were transiently transfected with HRP constructs using Lipofectamine 2000 reagent followed by irradiation treatment with 0 or 6 Gy. Western blot analysis of HRP protein was conducted after a 24-hour incubation. Cells were lysed, separated on 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gels, and transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The blocked membranes were incubated with a primary antibody against HRP (Bioss, Woburn, Mass) and a secondary HRP-conjugated goat antirabbit immunoglobulins (Pierce, Rockford, Ill). Immunoreactive proteins were observed using the enhanced chemiluminescent detection. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein loading control. The results were analyzed with ImageJ software (National Institutes of Health, Bethesda, Md).

Reverse Transcriptase-Polymerase Chain Reaction Analysis

Cells were transiently transfected with HRP-constructs using Lipofectamine 2000 reagent followed by irradiation with 0 or 6 Gy. Total RNA was isolated from cells using TRI Reagent (MRC Inc., Cincinnati, Ohio), and first-strand cDNA was synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas Inc., Hanover, Md) according to the manufacturer's instructions after a 24-hour incubation. The HRP cDNA was amplified for 35 cycles (1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C) using the primers 5′CATTCGGGAACGCTAACA3′ (forward) and 5′CGGACAGAGCCACAAG GT 3′ (reverse). The amplification of GAPDH cDNA was selected as an internal control. The polymerase chain reaction (PCR) products were resolved on 2.5% agarose gels and stained with ethidium bromide (Sigma, St. Louis, Mo). Gel images were obtained by using the Genesnap gel imaging system, and the densities of PCR products were quantified using Genetools gel analysis software (Syngene Bio Imaging, Cambridge, United Kingdom). All experiments were repeated at least 3 times.

Clonogenic Assays

For assessment of GDEPT, cancer cells transiently transfected with HRP constructs, were plated at low density in 25-cm2 flasks, left to settle over night, and exposed to culture medium containing either 0.5 mM IAA or buffer only for 24 hours under standard conditions. Cells were then irradiated in flasks with doses of 0 to 10 Gy of 60Co γ-rays at room temperature and were cultured under standard conditions for 14 days to allow for colony formation. After fixation and staining with 1% (weight/volume) crystal violet (Sigma) in dehydrated alcohol, colonies of >50 cells were scored, and surviving fractions were normalized for the plating efficiency of mock-irradiated cells. Survival curves were analyzed according to the linear-quadratic model, and the sensitizer enhancement ratio (SER) was assessed from the survival curves as the ratio of radiation doses that reduced cell survival by 50%: SER indicates the ratio of radiation dose at 50% survival fraction of transfected cells exposed to buffer and that of transfected cells exposed to IAA.

Apoptosis Assays

For apoptosis assays, cancer cells were transiently transfected with HRP constructs and divided into 4 groups after 24-hours: 1) the control group (no any treatment), the IR group (6-Gy irradiation only), 3) the drug group (0.5 mM IAA), and 4) the drug and IR group (combined IAA and 6-Gy irradiation). Apoptosis rates for the 3 different groups in 3 types of cancer cells were quantified by staining cells with both annexin V-fluorescein isothiocyanate (FITC) (PharMingen, San Diego, Calif) and propidium iodide (PI) (PharMingen) following the manufacturer's instructions and then analyzed with fluorescence-activated cell sorting flow cytometry (FACScan; Beckman Cytomics, Brea, Calif). Annexin V-FITC-positive/PI-negative cells were scored as early apoptotic, and annexin V-positive/PI-positive cells correspond to late apoptotic cells.

Construction of Adenovirus

The selected promoters were cotransformed with pAdEasy-1 into BJ5183 Escherichia coli cells using electroporation methods. After selection on kanamycin plates, positive colonies were selected and screened for recombinants by plasmid size and restriction enzyme analysis. The package, amplification, and purification of the recombinant adenovirus were accomplished by Shanghai GeneChem Co., Ltd. (Shanghai, China). The recombinant adenovirus was stored at −80°C until use. The negative control adenovirus (Ad-CMV-green fluorescent protein [GFP]) was purchased from Shanghai GeneChem Co., Ltd.

Animal Experiments

BALB/C nude mice aged 4 to 6 weeks purchased from Shanghai Experimental Animal Center (Chinese Academy of Sciences, Shanghai, China). The MHCC97 cell line was selected in the xenograft mouse model because of its higher tumorigenicity in nude mice compared with the other 2 cancer cell lines. MHCC97 cells were injected subcutaneously into the left dorsal leg of the mice. After 10 days, the average diameter of ectopic xenograft tumors reached 3 to 5 mm, and 48 mice were divided randomly into 4 groups: Ad-CMV-GFP, Ad-C6-hTERT-HRP, Ad-CMV-GFP plus IR, and Ad-C6-hTERT-HRP plus IR. The mice were given intratumoral injections of 1 × 109 plaque-forming units of viruses on the 11th, 14th, and 17th days postinoculation followed by 6-Gy irradiation treatment on the next day in the IR-plus groups. All mice were injected intraperitoneally with IAA (5 mM, 100 μL per mouse) from the 13th to 27th days. Tumor volumes were estimated every week according the following formula: relative volume = 0.5 × length × width.2 The survival time of mice was estimated, and the endpoint of observation was at the end of the eighth week. The tumors and normal tissues from each mouse were taken for hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate labeling (TUNEL) assay at the end of the experiment. Apoptotic cells were counted under a light microscope in randomly chosen fields, and the apoptosis rate was calculated as the percentage of apoptotic cells that scored ≥1000.

Statistical Analysis

All numerical experimental data were expressed as means ± standard deviations, and statistical analyses of data were performed using analysis of variance and t test methods. Survival analysis of mice was assessed by using the Kaplan-Meier Method. All P values are based on 2-sided hypothesis testing, and P < .05 is considered significant.

RESULTS

Radiation Inducibility of Synthetic Promoters

All chimeric promoters that we constructed in this study were subjected to a dual-luciferase reporter assay. Similar results were observed in the 3 cancer cell lines A549, MHCC97, and HeLa, as indicated in Figure 1: The C4-hTERT, C6-hTERT, C8-hTERT promoters all had radiation-inducible responses compared with the single-hTERT promoter, and nearly maximum levels were obtained at 4 to 6 Gy. The C6-hTERT promoter was identified as more inducible than the C4-hTERT and C8-hTERT promoters (activity levels: 16.11 ± 0.622 vs 12.36 ± 2.63 and 7.683 ± 3.615, respectively, at 6 Gy in HeLa cells; 2.297 ± 0.615 vs 1.563 ± 0.261 and 1.088 ± 0.193, respectively, at 6 Gy in A549 cells; and 5.312 ± 0.715 vs 3.75 ± 1.037 and 2.735 ± 1.594, respectively, at 6 Gy in MHCC97 cells; P < .05), but no radiation inducibility could be detected with the C10-hTERT promoter. In normal hEL cells, very low activity was observed in the single-hTERT, C4-hTERT, C6-hTERT, C8-hTERT, and C10-hTERT promoters compared with controls (activity levels: 0.674 ± 0.285, 0.787 ± 0.153, 1.025 ± 0.115, 0.82 ± 0.083, 0.614 ± 0.282, and 2.213 ± 0.221, respectively, at 6 Gy in hEL cells; P < .05).

Figure 1.

Radiation-mediated activation of gene promoters is illustrated. The relative luciferase (luc) activity after various doses of irradiation was measured on an illuminometer using the Dual Luciferase Reporter Assay System (Promega, Madison, Wis). Statistical analyses of data were performed using analysis-of-variance methods. hTERT indicates human telomerase reverse transcriptase; Gy, grays.

Detection of HRP Expression

The vector C6-hTERT-HRP was constructed for cancer GDEPT, and hTERT-HRP, control-HRP, and control-Luc were used as single promoter, positive, and negative controls, respectively. Cells were transiently transfected with these vectors followed by Western blot analysis to determine the expression of HRP protein. Detectable proteins were investigated in the 3 cancer cell lines transfected with control-HRP, C6-hTERT-HRP, and hTERT-HRP (Fig. 2, top); whereas no proteins could be detected in luciferase-expressing cells. After 6-Gy radiation treatment, higher HRP expression of the chimeric promoter could be detected in all 3 cancer cell lines (0.89 ± 0.08 vs 1.54 ± 0.12 in HeLa cells, 0.9 ± 0.05 vs 2.2 ± 0.1 in A549 cells, and 0.80 ± 0.09 vs 1.72 ± 0.05 in MHCC97 cells; P < .05) (Fig. 2, bottom). IR-inducible effects of HRP messenger RNA (mRNA) expression were confirmed by reverse transcriptase-PCR (RT-PCR). The increase in expression of HRP mRNA controlled by the C6-hTERT promoter was promoted significantly when cancer cells were exposed to a radiation dose of 6 Gy, as demonstrated in Figure 3 (2.16-fold increase in HeLa cells, 1.32-fold in A549 cells, 1.33-fold in MHCC97 cells), and no significant increase was detected in cells that were controlled by the control promoter or the hTERT promoter (0.997-fold and 1.05-fold in HeLa cells, respectively; 0.66-fold and 0.98-fold in A549 cells, respectively; and 1.02-fold and 1.07-fold in MHCC97 cells, respectively). In normal cells, scant HRP proteins and HRP mRNA expression were detected in C6-hTERT-HRP, and no IR-inducible effect was observed.

Figure 2.

The detection of horseradish peroxidase (HRP) protein expression is illustrated. The cancer cell lines HeLa, A549, and MHCC97 along with normal human embryonic lung (hEL) cells were transiently transfected with plasmid constructs, and the expression of HRP protein was detected by Western blot analysis. (Top) Representative HRP protein expression is shown: lane 1, control-HRP; lane 2, control-HRP and 6-gray (Gy) trigger; lane 3, the promoter carrying 6 tandem-repeat copies of the prototype CArG sequence (C6) combined with human telomerase reverse transcriptase (hTERT) and HRP (C6-hTERT-HRP); lane 4, C6-hTERT-HRP and 6-Gy trigger; lane 5, hTERT-HRP; lane 6, hTERT-HRP and 6-Gy trigger. (Bottom) Results from HRP expression analyses are shown. All numerical data were analyzed statistically using t tests.

Figure 3.

The detection of horseradish peroxidase (HRP) messenger RNA (mRNA) expression is illustrated. The cancer cell lines HeLa, A549, and MHCC97 and normal human embryonic lung cells (hEL) were transiently transfected with plasmid constructs, and the expression of HRP mRNA was detected by reverse transcriptase-polymerase chain reaction analysis. (Top) Representative HRP mRNA expression is shown: lane 1, human telomerase reverse transcriptase (hTERT)-HRP; lane 2, hTERT-HRP and 6-gray (Gy) trigger; lane 3, the promoter carrying 6 tandem-repeat copies of the prototype CArG sequence (C6) combined with hTERT-HRP; lane 4, C6-hTERT-HRP and 6-Gy trigger; lane 5, control-HRP; lane 6, control-HRP and 6-Gy trigger. (Bottom) Results from HRP mRNA expression analyses are shown. All numerical data were analyzed statistically using t tests.

GDEPT Assays for Constructed Vectors

The therapeutic potential of chimeric promoter-mediated gene therapy combined with radiotherapy was analyzed using clonogenic and apoptosis assays. Survival curves for the chimeric promoter system in different cancer cell lines are provided in Figure 4. HRP-transfected cancer cells that were exposed to IAA had increased cell kill levels compared with cells that were exposed to buffer only, whereas no sensitization was induced in luciferase-expressing cells. The SER was calculated as the ratio of the radiation dose at a 50% survival fraction of transfected cells exposed to buffer and that of transfected cells exposed to IAA, and the SER values for different vectors are listed in Table 1. The C6-hTERT promoter system was more sensitive to irradiation than the hTERT and control promoters (P < .05). The enhancement of apoptosis induction in cancer cells was detected by annexin V staining. Figure 5 indicates that the mean percentage of apoptotic cells in the drug and IR group was significantly increased compared with control, drug, or irradiation group in all 3 cancer cell lines except in luciferase-expressing cells. The combination of C6-hTERT-HRP/IAA with 6-Gy radiation resulted in a significantly higher apoptotic level (53.8% ± 3.3% in HeLa cells, 43.7% ± 4.4% in A549 cells, and 36.6% ± 3.2% in MHCC97 cells) compared with hTERT-HRP (22.1% ± 1.1% in HeLa cells, 20.6% ± 2.4% in A549 cells, and 18.6% ± 2.3% in MHCC97 cells) or control-HRP (29.5% ± 9.5% in HeLa cells, 32.2% ± 3.6% in A549 cells, and 22.9% ± 2.4% in MHCC97 cells; P < .05) in the drug and IR group.

Figure 4.

These survival curves of the chimeric promoter system in the cancer cell lines (A) HeLa, (B) A549, and (C) MHCC97 were obtained and analyzed with Graphpad Prism4 statistical software (GraphPad Software, Inc., San Diego, Calif). Squares indicate the promoter carrying 6 tandem-repeat copies of the prototype CArG sequence (C6) combined with human telomerase reverse transcriptase and horseradish peroxidase (C6-hTERT-HRP) plus the prodrug indole-3-acetic; triangles, C6-hTERT-HRP only.

Figure 5.

Apoptotic analyses of cancer cells transfected with horseradish peroxidase (HRP) constructs in different groups are illustrated. Apoptosis rates from 3 different groups in 3 types of cancer cells were quantified by staining cells with both annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) and were then analyzed using fluorescence-activated cell sorting flow cytometry. (Left) These representative plots illustrate the chimeric promoter system in different cancer cells. (Right) The percentages of apoptotic cells in different groups are shown. Statistical analyses of data were performed using analysis-of-variance methods. IR indicates ionizing radiation; hTERT, human telomerase reverse transcriptase; HRP, horseradish peroxidase; C6, the promoter carrying 6 tandem-repeat copies of the prototype CArG sequence; luc, luciferase.

Table 1. Values of Sensitizer Enhancement Ratios for Different Vectors in 3 Cancer Cell Linesa
 Cell Line: Mean±SDb
VectorHeLA549MHCC97
  • Abbreviations: HRP, horseradish peroxidase; Luc, luciferase; pC6, promoter carrying 6 tandem-repeat copies of the prototype CArG sequence; pControl, control promoter; phTERT, human telomerase reverse transcriptase promoter; SD, standard deviation.

  • a

    The sensitizer enhancement ratio (SER) was calculated as SER=radiation dose at 50% survival fraction in plasmid group/radiation dose at 50% survival fraction in the plasmid and indole-3-acetic acid group.

  • b

    The means±SD of at least 3 independent experiments are shown.

pControl-Luc1.01±0.261.08±0.090.98±0.15
phTERT-HRP1.64±0.342.43±0.141.85±0.22
pC6-hTERT-HRP3.18±0.373.42±0.133.2±0.46
pControl-HRP2.21±0.252.88±0.312.47±0.19

Effect of Adenovirus-Mediated Gene Therapy in a Mouse Model

The therapeutic efficacy of the combination of adenovirus-mediated suicide gene therapy plus IR was demonstrated in vivo in a human hepatocellular carcinoma xenograft mouse model. Figure 6A reveals that the combination of adenovirus-mediated gene therapy and radiotherapy significantly inhibited tumor volume compared with the adenovirus group. Furthermore, the relative tumor volume in the Ad-C6-hTERT-HRP plus IR group was significantly smaller than that in the Ad-CMV-GFP plus IR group 5 weeks after inoculation (P < .05). Survival curves for the mice are provided in Figure 6B. Mice in the Ad-C6-hTERT-HRP plus IR group had significantly prolonged survival compared with all other groups (P < .05). The mean survival duration was 54.2 days in the Ad-C6-hTERT-HRP plus IR group, 30.5 days in the Ad-CMV-GFP group, 47.7 days in the Ad-C6-hTERT-HRP group, and 46.7 days in the Ad-CMV-GFP plus IR group. Figure 7 provides the results from TUNEL staining in these tumors. The combination of Ad-C6-hTERT-HRP and radiotherapy resulted a higher apoptotic level (24.73% ± 3.71%), compared with the Ad-C6-hTERT-HRP group (9.56% ± 2.75%), the Ad-CMV-GFP and IR group (10.92% ± 2.43%), and the Ad-CMV-GFP group (1.08% ± 0.55%; P < .05). Normal tissues (heart, liver, and kidney) were collected for H&E staining at the end of the experiment. In general, these tissue samples had no obvious changes observed under a microscope, and no toxicity effect associated with treatment on normal tissues could be detected (Fig. 8).

Figure 6.

The effect of combined adenovirus (Ad)-mediated gene therapy with radiotherapy is illustrated in a xenograft mouse model. (A) The average tumor volume per mouse is illustrated in different groups. Data on tumor volumes and apoptosis rates were analyzed using analysis-of-variance methods. (B) Survival curves are shown for mice in different groups. Survival analysis of mice was assessed by using the Kaplan-Meier method. CMV indicates cytomegalovirus; GFP, green fluorescent protein; IR, ionizing radiation; hTERT, human telomerase reverse transcriptase; HRP, horseradish peroxidase.

Figure 7.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of tumor tissues reveals (A) the group transfected with adenovirus (Ad) with a new promoter carrying 6 tandem-repeat copies of the prototype CArG sequence (C6) combined with human telomerase reverse transcriptase (hTERT)-horseradish peroxidase (HRP), (B) the Ad-C6-hTERT-HRP and ionizing radiation (IR) group, (C) the Ad cytomegalovirus promoter-green fluorescent protein (CMV-GFP) group, and (D) the Ad-CMV-GFP and IR group.

Figure 8.

These are photomicrographs of normal hematoxylin and eosin-stained tissues from the (A) heart, (B) liver, and (C) kidney.

DISCUSSION

Studies have demonstrated that some suicide gene systems, like the herpes simplex virus thymidine kinase and ganciclovir system,18 can radiosensitize cells, resulting in increased radiation cytotoxicity for a given dose. In the current study, rational strategies for combing a HRP/IAA system controlled by novel chimeric gene promoters with radiotherapy were explored. There are some advantages to this chimeric promoter system for cancer therapy: First, the combination of HRP/IAA gene therapy with ionizing radiation presents possible therapeutic advantages in the treatment of solid malignancies, in particular to target the hypoxic population.19, 20 Second, combined gene therapy and radiotherapy protocols have the potential to overcome many of the limitations of dose delivery in radiotherapy. The combination of gene therapy strategies with radiotherapy may increase the effectiveness of each radiation dose fraction, resulting in a lower dose, reduced normal tissue damage, and improved patient quality of life.21 Third, CArG elements also can enhance the activity of hTERT promoter through irradiation but do not dampen its specificity to cancer cells. In particular, radiation can be exploited to selectively activate gene expression in the tumor mass.

Some researchers have used promoters of radiation-inducible genes to drive the transcription of transgenes in the response to radiation. For example, the genetic elements in the Egr-1 promoter that control the response to radiation were delineated in a series of experiments, and radiation inducibility was pinpointed to 10 nucleotide motifs of consensus sequence known as CArG elements.22 The precise mechanism of this response and the transcription factors that interact with CArG elements have yet to be elucidated. However, it is known that complexes of phosphorylated transcription factors and accessory proteins, including serum response factor (SRF), promote gene expression by binding to CArG motifs.23 Egr-1 enhancer deletion studies also have demonstrated that multiple CArG elements confer greater promoter inducibility than a single element to both SRF and irradiation.24 In the current study, we demonstrated that increasing the numbers of CArG elements from 4 to 6 improves the induction response to irradiation, which may be explained simply because more target sequences are provided for the efficient binding of specific transcription factor complexes. However, when 8 CArG elements were used, the response was no more than that for 4 and 6 elements, and no irradiation-inducible response was detected with 10 CArG elements. Thus, the continual addition of CArG elements may not necessarily increase enhancer irradiation response. Another research team has revealed that increasing the number of CArG elements up to a certain level (9 CArG elements) increased the CMV promoter radiation response14; however, our results revealed little difference in the level of response compared with the number of CArG elements used. This maybe because the specific hTERT promoter resulted in different gene expression. Some crystallographic studies have indicated that the binding of SRF to CArG sequences causes “bending” of the DNA helix as it wraps around the protein.25 Cumulative changes in secondary structure caused by the occupancy of multiple binding sites may not present a suitably accessible configuration and eventually may lead to the down-regulation of gene expression. It is noteworthy that the saturation effect of responsive element number also has been reported in several studies. For instance, the insertion of 5 copies of hypoxia-responsive elements derived from the vascular endothelial growth factor promoter resulted in maximal enhancement of hypoxia responsiveness.26

To determine whether the chimeric promoter-controlled gene expression was applicable to cancer therapy, plasmid vectors encoding the enzyme HRP were constructed, and we detected the radiation-inducible effect of HRP expression driven by the chimeric promoter using Western blot and RT-PCR analyses. A significant increase in HRP protein expression was detected in cancer cells that were transfected with C6-hTERT-HRP after treatment with 6-Gy irradiation. However, the increased mRNA expression level maybe not coincident with the change observed at the protein level. This may be caused by the low sensitivity of RT-PCR. Cancer cells were transiently transfected with these constructs, and their sensitivity to the prodrug IAA was examined. The plasmid that contained chimeric promoter produced higher cell kill levels than the levels observed with hTERT-HRP and produced even higher levels compared with control-HRP, demonstrating the potential of these systems for future use. Higher HRP expression driven by the chimeric promoter compared with the hTERT and control promoters may be the major reason, as illustrated in Figures 2 and 3, and the increased toxic effects in cancer cells are illustrated in Figures 4 and 5. Apoptosis is known as 1 of the important mechanisms of cytotoxicity induced by the HRP/IAA system. HRP/IAA selectively induced exposure of phosphatidylserine, DNA fragmentation, and chromatin condensation in treated cells, and caspases appeared to be involved as an “effector” in the apoptosis process.27 Another study also indicated that IAA/HRP reaction leads to caspase-3 activation and poly-(ADP-ribose) polymerase (PARP) cleavage, which can be blocked by catalase, and IAA/HRP-produced hydrogen peroxide, which may be an important mediator of IAA/HRP-induced apoptotic cell death.28 In the current study, we demonstrated that the HRP/IAA system controlled by chimeric promoters can induce a significant increase in apoptotic cells compared with controls. Cells that initiate apoptosis cannot be clonogenic, yet the percentages of apoptotic cells detected cannot fully account for the killing levels measured with the clonogenic assay. Several investigators have observed that cell sensitivity as assessed by clonogenic ability is greater than that measured by apoptosis assays.28 Indeed, PI staining of unfixed cells suggests that some cells undergo necrotic death, which also contributes to the lost of their clonogenic ability. Moreover, apoptosis is a dynamic process and may be underestimated by examining cells at a certain time point. In particular, at the time of detection, the cells do not appear to have attempted to undergo mitosis yet, and delayed apoptosis may be occurring.

We also tested the antitumor effect of this chimeric promoter system mediated by adenovirus vector in a mouse xenograft model. The chimeric promoter system revealed higher antitumor activity compared with controls, and no toxicity effect associated with the treatment could be detected. Mice in the Ad-C6-hTERT-HRP plus IR group also had longer survival compared with controls. A recent study characterized the HRP/IAA system in vivo for gene therapy and indicated that HRP-expressing tumors had a modest growth delay when treated with IAA compared with drug vehicle controls, but the treatment response could not be improved by combining HRP-directed gene therapy with fractionated radiotherapy.29 However, in the current study, we demonstrated that the combination of gene therapy mediated by chimeric promoters with radiotherapy increased the antitumor effect. These different results may have been because 1) radiation increases the expression of HRP through chimeric promoters and, hence, increases in the GDEPT effect; or 2) different cancer cells may have a different response to irradiation and gene therapy.

The results presented here demonstrate that cancer-specific and radiation-inducible promoters offer the potential to selectively control therapeutic gene expression in tumors while minimizing normal tissue toxicity. The chimeric hTERT promoter containing 6 repeat CArG elements had the best radiation response compared with other chimeric promoters that contained different numbers of CArG elements. GDEPT assays with the synthetic promoters agreed with the results of the Luc reporter assays. Both in vitro and in vivo studies indicated showed that the combinational promoter system has the potential for use in cancer gene therapy. In conclusion, the high-level, cancer-specific gene expression system controlled by irradiation can represent an interesting and promising approach for future clinical application.

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FUNDING SOURCES

This study was supported by the National Natural Science Foundation of China (Grant 30672438), the Hubei Provincial Natural Science Foundation of China (Grants 2006ABC009 and JX4A06), and the Wuhan City Science and Technology Foundation (Grants 200960323129).

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

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