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Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model
Biochemical and Biophysical Research Communications 412 (2011) 763–768
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model Yu Xu a,b, Zhengchun Liu b, Haiyan Kong b, Wenjie Sun a,b, Zhengkai Liao a,b, Fuxiang Zhou a,b, Conghua Xie a,b, Yunfeng Zhou a,b,⇑ a b
Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, PR China Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071, PR China
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Article history: Received 13 August 2011 Available online 22 August 2011 Keywords: Gene therapy hTERT promoter CArG element c-Irradiation HRP/IAA system Interleukin 12
a b s t r a c t The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Gene therapy is a novel approach for cancer treatment, which has been evaluated in a great deal of preclinical and clinical trials over the past years. However, side effects due to the lack of tumor specificity limited its application. Therefore, targeted gene therapy strategies were developed to prevent the toxicity of therapeutic gene in normal cells, cell- or tissue-specific promoter being the major one [1]. The human telomerase reverse transcriptase (hTERT) promoter which is highly activated in cancer cells but not in normal differentiated cells has been widely used to drive the expression of therapeutic genes in various cancer cells [2–4]. However, the transcriptional activity of hTERT promoter is weaker than the nonspecific cytomegalovirus (CMV) promoter that is commonly used, resulting in insufficient therapeutic gene expression [5]. Hence, additional regulatory strategies are needed to increase the transcriptional activity of the hTERT promoter.
⇑ Corresponding author at: Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, PR China. Fax: +86 27 67812889. E-mail addresses: [email protected] (Y. Xu), [email protected] (Z. Liu), [email protected] (H. Kong), [email protected] (W. Sun), fastbeta@ gmail.com (Z. Liao), [email protected] (F. Zhou), [email protected] (C. Xie), [email protected] (Y. Zhou). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.077
The early growth response-1 (Egr-1) promoter has been described as a radiation-inducible promoter, whose activation depends on CArG elements, a 10 nucleotide motifs of consensus sequence CC(A/T)6GG found in the promoter region of Egr-1 gene [6,7]. In order to reduce the nonspecific binding sites of the Egr-1 promoter, a novel chimeric promoter consisting of tandem repeats of isolated CArG elements and the CMV promoter was designed to drive suicide gene expression, which showed better radiation inducibility than the wild-type Egr-1 promoter [8,9]. In the study, we designed a novel dual radiation-inducible and tumor-specific promoter system by combining CArG elements and the hTERT promoter, which can target only to cancer cells as well as sufficient transcriptional activity. To test the potential clinical benefit of such an approach, a gene-directed enzyme prodrug therapy (GDEPT) system consisting of the horseradish peroxidase (HRP) gene driven by the chimeric promoter and the prodrug indole-3-acetic (IAA) was developed and its antitumor effect was evaluated in vitro and in vivo. Furthermore, cytokine-based immuno-gene therapy has been shown to be involved in the process of suicide gene therapy [10]. Interleukin 12 (IL12) is a heterodimeric cytokine that plays multiple roles in the immune system such as augmenting the proliferation and cytotoxic activity of T cells and NK cells [11]. Our previous study showed that the combination of HRP/IAA system and IL12 had cumulative antitumor effects [12]. Therefore, we also considered that adenovirus-mediated IL12 gene could enhance
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Fig. 1. Construction and characterization of synthetic promoter with tumor specificity and radiation inducibility. (A) Schematic drawing of the construction for the chimeric promoters. Luciferase gene was driven by the chimeric promoter consisting of repeated CArG elements (n = 4, 6, 8 or 10) and the hTERT promoter. (B) Tumor specificity of synthetic promoters. Luciferase reporter plasmids containing various promoters were introduced into LLC cells and MRC-5 cells which were telomerase- positive and negative, respectively. (C and D) Radiation inducibility of synthetic promoters. Transfected LLC cells were exposed at 4-Gy irradiation to screen for the optimal number of repeated CArG elements (C), and then treated with various doses (0, 2, 4, 6, 8, 10 Gy) of radiation to obtain the most inducible dose (D). The data of luciferase assay were expressed as relative activity with regard to pGL3-control, which was considered as 100%. Data represent means ± SD of at least three independent experiments.
the antitumor effect of HRP/IAA system driven by the novel chimeric promoter in a murine model of Lewis lung carcinoma (LLC). 2. Materials and methods 2.1. Cell culture LLC cell line and human MRC-5 cell line were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and maintained in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml streptomycin. Cells were incubated at 37 °C in an atmosphere containing 5% CO2. All culture reagents were purchased from Hyclone (Logan, UT, USA). 2.2. Animals Female C57BL/6 mice (6–8 weeks) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and housed in a specific pathogen-free (SPF) facility at the Animal Experimental Center of Wuhan University. The facilities and the protocol of this experiment were consistent with the regulations on animal use for biomedical experiments issued by the Ministry of Science and Technology of China and approved by the Animal Care Committee of Wuhan University.
phTERT-luc) as we described previously [13]. The synthetic enhancers containing different numbers of CArG elements were cloned as double-stranded linker molecules derived from complementary single-stranded oligonucleotides: 50 -CGCGTGATAT(CCTTA TTTGG)n-30 and 50 -AATT(CCAAATAAGG)nATATCA-30 (n = 4, 6, 8, 10). The synthetic enhancers were placed upstream of the hTERT promoter in phTERT-luc by using the MulI and EcoRI sites at the linker ends. This created four plasmids containing luciferase gene driven by chimeric promoters consisting of CArG elements and the hTERT promoter, which were pC4-hTERT-luc, pC6-hTERT-luc, pC8-hTERTluc and pC10-hTERT-luc, respectively. Schematic drawing of the construction for the chimeric promoters is available in Fig. 1A. 2.4. Luciferase assay The expression of the luciferase gene driven by the chimeric promoter was determined using the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instruction. Briefly, cells plated in 24-well culture plate at 80– 90% confluency were transfected with 0.4 lg of the luciferase reporter plasmid and 0.4 lg of the pRL-TK plasmid. Twenty four hours later, cells were irradiated using a 60Co gamma (c)-ray source (GWXJ80 type, Chengdu, China) and analyzed for radiation-induced gene expression by luciferase assay after another 24 h. The pRL-TK plasmid was used as an internal control. The simian virus 40 (SV40) promoter (pGL3-control) was used as a positive control and its luciferase activity was considered to be 100%.
2.3. Vector construction 2.5. Adenovirus construction The pGL3-basic plasmid (Promega, Madison, USA) was used as the basis for all the vector constructs. The hTERT promoter was placed upstream of the luciferase gene in pGL3-basic (creating
All adenovirus vectors were constructed using AdEasy System as we described previously [14]. Briefly, virus vectors expressing
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HRP gene driven by a chimeric promoter (AdC6-hTERT-HRP) and murine IL12 (mIL12) gene under the control of the CMV promoter (Ad-CMV-mIL12) were constructed by homologous recombination in Escherichia coli. Viruses were purified by double cesium chloride gradient ultracentrifugation. Viral tilter was determined by plaque assay and expressed as plaque forming unit (pfu). Purified virus aliquots were stored at 80 °C. 2.6. Western blotting LLC cells were infected with adenoviruses expressing HRP at multiplicity of infection (MOI) of 1000. This MOI was selected as previous experiments had demonstrated a maximal level of transduction efficiency using these experimental protocols [12]. Twenty four hours later, cells were irradiated and then incubated for another 24 h. HRP expression in treated LLC cells was determined by Western blotting. The mouse anti-HRP antibody (dilution, 1:500) and mouse anti-b-actin (dilution, 1:1000) were used to detect corresponding proteins. All the antibodies including goat antimouse secondary antibody (dilution, 1:10,000) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). 2.7. Cytotoxicity of HRP/IAA system in vitro Transduced LLC cells (2 103/well) were plated in 96-well culture plates and irradiated 24 h later, followed by IAA (Sigma, MO, USA) administration at a concentration of 0–5 mM. Media containing different concentration of IAA were exchanged every 48 h. Cell viability was determined using MTT assay (Invitrogen, CA, USA) 120 h later and optical density values were measured by a microplate reader (Turner BioSystems, CA, USA). 2.8. In vivo antitumor effects A total of 5 106 LLC cells were injected subcutaneously in the right flank of C57BL/6 mice. Tumors were isolated, prepared into cell suspension and inoculated into new mice 14 days later. When tumors reached 5 mm in diameter (day 10), the mice were randomized to treatment groups of 10 mice: Ad-CMV-GFP group, Ad-CMV-mIL12 group, AdC6-hTERT-HRP + irradiation (Trigger) group and combination group (AdCMVmIL-12 + AdC6-hTERTHRP + Trigger). The mice were treated with 1 109 pfu Ad-CMVGFP, 5 108 pfu Ad-CMV-mIL12 or 5 108 pfu AdCx-hTERT-HRP adenoviruses diluted in 30 ll PBS by intratumoral administration. Local c-irradiation (Trigger) was performed 24 h later (day 11). IAA (50 mg/kg daily) was administered to the mice by intraperitoneal injection for seven consecutive days from day 12 to 18. Twenty four hours later, five mice of each group were sacrificed for histological examination and toxicity study. The remaining mice were examined every 3 days for evaluation of tumor growth. Tumor sizes were measured using a caliper in mm3 and volumes were calculated using the following formula: (L W2)/2, where L equals length and W equals width. Study endpoint was death or sacrifice of the animal with very high tumor volume that exceeded 3500 mm3. 2.9. Immunohistochemistry Tumor tissues were fixed using formalin and 4 lm sections were stained with hematoxylin and eosin for routine histological analysis. For immunohistochemical analysis, fresh-frozen acetone-fixed sections were stained for infiltration T lymphocytes (CD4+ and CD8+) with specific antibodies (BD PharMingen, CA, USA) using standard peroxidase method. Positive staining was scored by light microscopy. After initial scanning under 100 magnification, positively stained cells in 10 fields under 400 magnifi-
Fig. 2. Expression (A) and biological activity (B) of exogenous HRP in vitro. LLC cells were infected with Ad-CMV-GFP and AdC6-hTERT-HRP with or without triggering by 6-Gy irradiation. (A) HRP expression in transduced LLC cells was detected by Western blotting. (B) To evaluate the biological activity of exogenous HRP, cytotoxicity was determined using MTT assay after IAA administration at indicated concentrations in transduced LLC cells. Data were expressed as percentage relative to drug-free cells. Each point represented the means ± SD of three independent experiments.
cation were counted and the mean number/high power field (HPF ± SEM) was determined. 2.10. In vivo toxicity studies Biochemistry parameters including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in the sera from treated animals were measured to evaluate side effects of treatment. Key organs and tissues were harvested, fixed with formalin and stained with hematoxylin and eosin for histological analysis. Three mice without any treatment were used as normal control. 2.11. Statistical analysis The significance of differences between experimental groups was calculated using Student’s t-test or one-way ANOVA analysis as appropriate. Statistical analyses were performed with SPSS software version 13.0 (SPSS Inc. Chicago, IL) and two-tailed P < 0.05 was considered as statistically significant.
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3. Results 3.1. Characterization of synthetic promoters To evaluate the specificity of chimeric promoters, LLC cells and MRC-5 cells were transfected with various plasmids constructed. As shown in Fig. 1B, the hTERT promoter and synthetic promoters showed higher activity in LLC cells than in MRC-5 cells (P < 0.01). Then transfected LLC cells were treated with 4-Gy c-irradiation to evaluate the inducibility of chimeric promoters (Fig. 1C). The results showed that the C6-hTERT promoter was more inducible than other promoters (C6-hTERT vs hTERT, 1.38-folds, P = 0.000; C6hTERT vs C4-hTERT, 1.19-folds, P = 0.000; C6-hTERT vs C8-hTERT, 1.18-folds, P = 0.001; C6-hTERT vs C10-hTERT, 1.28-folds, P = 0.000). LCC cells were transfected with pC6-hTERT-luc and treated with various doses (0, 2, 4, 6, 8, 10 Gy) of c-irradiation (Fig. 1D). It was evident that the level of luciferase expression observed was dependent on radiation dose, with maximal expression seen after 6 Gy (6 vs 0 Gy, 1.61-folds, P = 0.000; 6 vs 2 Gy, 1.45folds, P = 0.001; 6 vs 4 Gy, 1.2-folds, P = 0.030; 6 vs 8 Gy, 1.23-folds, P = 0.017; 6 vs 10 Gy, 1.45-folds, P = 0.001). Therefore, the pC6hTERT promoter and 6-Gy irradiation were chosen for the subsequent studies. 3.2. HRP expression in LLC cells in vitro
tumor growth was more inhibited in the combination group than treatment with Ad-hTERT-HRP alone without radiation (p = 0.001 on days 12–36) [12]. As assessed by microscopic examination, the therapeutic effects of combination strategy were more significant; the tumor nodules in combination groups contained larger necrotic areas than other groups (data not shown). The results indicate the potent potentials of the combination strategy for cancer treatment. 3.5. T lymphocytes infiltration in tumors To explore the mechanism of cumulative antitumor effects, the number and distribution of CD4+ and CD8+ T cells in tumor tissues were evaluated using IHC. As shown in Fig. 4A–H, both CD4 and CD8 were stained in the cell membrane of interstitial infiltrates. The combination group showed extensive infiltration of CD4+ T cells and CD8+ T cells in comparison with Ad-CMV-GFP group (CD4, 8.6-folds, P = 0.000; CD8, 8.1-folds, P = 0.000), Ad-CMVmIL12 group (CD4, 1.4-folds, P = 0.003; CD8, 1.5-folds, P = 0.000) and AdC6-hTERT-HRP (CD4, 1.9-folds, P = 0.000; CD8, 2.8-folds, P = 0.000) (Fig. 4I). 3.6. Toxicity studies in vivo To evaluate the toxicity of various strategies in vivo, biochemistry parameters of liver and kidney in sera and histological changes
LLC cells infected by Ad-CMV-GFP or AdC6-hTERT-HRP with or without 6-Gy irradiation treatment were harvested for determining HRP expression by Western blotting. The results showed that the HRP expression was observed in AdC6-hTERT-HRP transduced groups whereas not in Ad-CMV-GFP transduced groups (Fig. 2A). In addition, a significant increase in the HRP expression was observed after irradiation (1.69-folds, P = 0.000). These data suggest that the therapeutic genes were successfully delivered into the cells and the novel chimeric promoter resulted in increased transcriptional activity. 3.3. Cytotoxicity of the HRP/IAA system in vitro Transduced LLC cells were treated with IAA at indicated concentrations in Fig. 2B. The data were presented as percentages of cell viability with regard to control cells without IAA treatment. LLC cells in the AdC6-hTERT-HRP group and AdC6-hTERT-HRP + Trigger group exhibited a dose-dependent cytotoxicity, and the IC50 of IAA was about 3.0 and 1.5 mM, respectively. The results indicate that the HRP/IAA system controlled by chimeric promoter could be induced by radiation, which had sufficient cytotoxic effects on LLC cells. 3.4. Suppression of tumor growth in vivo To further evaluate the antitumor activity of AdC6-hTERT-HRP/ IAA and the cumulative antitumor effect of combining AdC6hTERT-HRP/IAA and Ad-CMV-mIL12 in vivo, we established an immunocompetent tumor model with LLC cells in C57BL/6 mice. The mice were assigned randomly to four groups (group 1, AdCMV-GFP; group 2, Ad-CMV-mIL12; group 3, AdC6-hTERTHRP + Trigger; group 4, Ad-CMV-mIL12 + AdC6-hTERT-HRP + Trigger). After IAA administration, the expression of HRP and mIL12 in the tumor tissues was detected by Western blot as shown in Fig. 3A. The tumor growth was monitored every 3 days (Fig. 3B). The data showed that the combination treatment with Ad-CMVmIL12 and AdC6-hTERT-HRP + Trigger resulted in remarkable inhibition of tumor growth in comparison with the other three groups (groups 4 vs 1, p = 0.034 on days 6–24; groups 4 vs 2, p = 0.046 on days 9–36; groups 4 vs 3, p = 0.000 on days 12–45). Moreover, the
Fig. 3. Tumor suppression in LLC mouse model. Subcutaneous LLC tumors were treated by intratumoral injection with Ad-CMV-GFP, Ad-CMV-mIL12 and AdC6hTERT-HRP + Trigger alone or in combination. (A) Expression of HRP and mIL12 in the tumor tissues were detected by Western blot. This result is representative of that obtained from three independent experiments. (B) Tumor volumes were monitored at the indicated time points after IAA administration. Each data point represented the mean tumor volume in that group. Error bars represented means ± SD.
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Fig. 4. Infiltration of CD4+ and CD8+ T lymphocytes in treated LLC tumors. Five treated mice of each group were sacrificed 24 h after last IAA administration. Tumor sections were analyzed by immunohistochemical staining using specific antibodies against CD4 (A–D) or CD8 (E–H). The positive cells were scored through light microscopy (I). After initial scanning under 100 magnification, positive stained cells in 10 filed under 400 magnification were counted and the mean number of stained cells was averaged over 10 fields.
of key tissues were examined for treated mice. The results of liver and kidney function tests of all treated animals were not statistically different from those of control animals (Table 1). Meanwhile, there were no marked pathological changes in heart, liver, spleen, lung and kidney of treated mice (data not shown). These data suggest that the treatment was well tolerated. 4. Discussion Gene therapy is among the approaches currently used to treat malignant tumors, which uses specific therapeutic genes that cause death in cancer cells. The adenoviral vector has been used as a transfer vehicle to introduce genes into tumor cells since it is more efficient than non-viral gene transfer methods [15]. In early attempts of gene therapy, therapeutic genes were driven by ubiquitous promoters such as the CMV promoter, which induce nonspecific toxicity to normal cells and tissues in addition to cancer cells [16]. Subsequently, novel tumor- and tissue-specific promoter systems have been developed to target tumor cells but not normal cells for reducing undesirable toxicity [17]. The promoter region of tumor specific hTERT was cloned and characterized, which was used to induce tumor-specific therapeutic gene expression in cancer gene therapy [18,19]. However, gene expression driven by the hTERT promoter is much lower than expression driven by commonly used viral promoters, such as CMV promoter and
Table 1 Serum biomarkers of liver and kidney function in each group (means ± SD). Group
ALT (U/L)
AST (U/L)
BUN (mg/dl)
Cr (lmol/L)
Normal Ad-CMV-GFP Ad-CMV-mIL12 AdC6-hTERT-HRP Combination F value P*
42.7 ± 2.70 45.3 ± 2.61 43.8 ± 2.38 44.7 ± 1.96 45.2 ± 1.73 0.350 0.841
124.9 ± 13.40 128.8 ± 9.91 130.4 ± 13.79 124.9 ± 9.68 129.8 ± 7.51 0.266 0.896
12.7 ± 2.00 13.8 ± 0.89 13.6 ± 0.82 12.7 ± 1.40 12.8 ± 1.43 0.806 0.537
6.7 ± 1.10 6.8 ± 0.47 7.2 ± 0.43 6.7 ± 0.74 7.1 ± 0.29 0.655 0.631
It was shown that there were no statistically significant differences in serum ALT, AST, BUN and Cr levels among different groups (P > 0.05). ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine. * P < 0.05 was considered to be statistically significant.
simian virus 40 (SV40) promoter [5]. It is necessary, then, to increase the activity of this promoter. In the current study, an attempt was made to maintain the tumor specificity of the hTERT promoter and to increase its activity by using a radiation-inducible CArG element. We constructed several synthetic hTERT promoters containing different number of CArG elements, which were introduced into hTERT positive (LLC) and negative (MRC-5) cells to show the difference in transcriptional activity between tumor and normal cells. The chimeric pro-
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moters showed higher transcriptional activity in LLC cells than in MRC-5 cells, suggesting the tumor specificity (Fig. 1B). Then the synthetic promoter with six repeated CArG elements was found to be more radiation-inducible than the others (Fig. 1C). Further study was performed to obtain the optimal dose that resulted in more induction, and the peak activity was found in cells treated with 6-Gy irradiation (Fig. 1D). Taking these data together, the C6-hTERT promoter triggered by 6-Gy irradiation showed the stronger transcriptional activity besides tumor specificity. Adenovirus vector containing the HRP gene under control of the C6-hTERT promoter was constructed and introduced into LLC cells. Using Western blot analysis (Fig. 2A), we detected HRP in AdC6hTERT-HRP infected LLC cells and the expression level was higher after triggering with 6-Gy irradiation, suggesting that therapeutic gene was successfully delivered by adenovirus vectors and the novel chimeric hTERT promoter increased the target gene expression. We further investigated the in vitro effects of HRP expression following IAA treatment and observed an IAA dose-dependent decrease in cell viability in LLC cells, which indicated the biological activity of exogenous HRP (Fig. 2B). Additionally, the AdC6hTERT-HRP + Trigger group showed the strongest cytotoxic effect at low concentration of IAA, suggesting that the increased HRP expression by the radiation via chimeric hTERT promoter could strengthen the killing effect of the HRP/IAA system. Delivery of cytokine genes to cancerous cells as cancer gene immunotherapy alters the local tumor environment and induces an anti-tumor immune response to facilitate tumor eradication [20]. We previously showed that the combination of HRP/IAA system and IL12 had cumulative antitumor effects [12]. In the study, we hypothesized that combining HRP/IAA system controlled by radiation-inducible and tumor-specific C6-hTERT promoter and IL-12 gene driven by the CMV promoter could produce more significant antitumor effects. To validate this hypothesis, we established an immunocompetent LLC murine model. As expected, the combination strategy showed stronger antitumor effects in comparison with single agent strategies (Fig. 3B). There are two possible explanations for the cumulative antitumor effects: increased infiltration of stimulated helper CD4+ T cells and cytolytic CD8+ T cells induced by the local expression of IL12 and further activation of the host antitumor immune response mediated by the enhanced cell killing effect of the HRP/IAA system driven by the chimeric promoter (Fig. 4). Although the recruitment of CD4+ and CD8+ T cells was lower, the triggered AdC6-hTERT-HRP vector inhibited tumor growth more than the Ad-CMV-mIL12 vector, which might be because the killing effects of the suicide gene system (including direct killing effect and by stander effect) were more directly and stronger than the immune-gene. Moreover, there were no pathological changes in the serum biomarkers of liver and kidney function (Table 1) and the histological examination of key organs, suggesting that the treatment was well tolerated. In this study, we designed a novel chimeric promoter which maintained the tumor specificity by hTERT promoter and increased the expression of therapeutic gene by the radiation-inducible element. The combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.
Acknowledgments We thank Dr. Lin Huang from Department of Pathology, University of Nebraska Medical Center and Dr. Jinxuan Hou from Department of Oncology, Zhongnan Hospital of Wuhan University to review the manuscript. This study was supported by Grants from the National Natural Science Foundation of China (No. 30672438), the Hubei Provincial Natural Science Foundation of China (Nos. 2006ABC009 and JX4A06) and the Scientific and Technological Project of Wuhan City (No. 200960323129). References [1] C. Wu, J. Lin, M. Hong, et al., Combinatorial control of suicide gene expression by tissue-specific promoter and microRNA regulation for cancer therapy, Mol. Ther. 17 (2009) 2058–2066. [2] J. Gu, S. Kagawa, M. Takakura, et al., Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers, Cancer Res. 60 (2000) 5359– 5364. [3] A.S. Majumdar, D.E. Hughes, S.P. Lichtsteiner, et al., The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters, Gene Ther. 8 (2001) 568–578. [4] D. Jacob, M. Bahra, G. Schumacher, et al., Gene therapy in colon cancer cells with a fiber-modified adenovector expressing the TRAIL gene driven by the hTERT promoter, Anticancer Res. 24 (2004) 3075–3079. [5] J.S. Song, Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer, Biosci. Biotechnol. Biochem. 68 (2004) 1634–1639. [6] M.A. Stackhouse, D.J. Buchsbaum, Radiation to control gene expression, Gene Ther. 7 (2000) 1085–1086. [7] R. Datta, E. Rubin, V. Sukhatme, et al., Ionizing radiation activates transcription of the EGR1 gene via CArG elements, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 10149–10153. [8] B. Marples, S.D. Scott, J.H. Hendry, et al., Development of synthetic promoters for radiation-mediated gene therapy, Gene Ther. 7 (2000) 511–517. [9] S.D. Scott, M.C. Joiner, B. Marples, Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy, Gene Ther. 9 (2002) 1396–1402. [10] S. Kuriyama, H. Tsujinoue, H. Yoshiji, Immune response to suicide gene therapy, Methods Mol. Med. 90 (2004) 353–369. [11] M.P. Colombo, G. Trinchieri, Interleukin-12 in anti-tumor immunity and immunotherapy, Cytokine Growth Factor Rev. 13 (2002) 155–168. [12] Y. Xu, J. Hou, Z. Liu, et al., Gene therapy with tumor-specific promoter mediated suicide gene plus IL-12 gene enhanced tumor inhibition and prolonged host survival in a murine model of Lewis lung carcinoma, J. Transl. Med. 9 (2011) 39. [13] Z.K. Liao, F.X. Zhou, Z.G. Luo, et al., Radio-activation of hTERT promoter in larynx squamous carcinoma cells: an ‘indirected-activator’ strategy in radiogene-therapy, Oncol. Rep. 19 (2008) 281–286. [14] J. Luo, Z.L. Deng, X. Luo, et al., A protocol for rapid generation of recombinant adenoviruses using the AdEasy system, Nat. Protoc. 2 (2007) 1236–1247. [15] Y.P. Zhang, L. Sekirov, E.G. Saravolac, et al., Stabilized plasmid–lipid particles for regional gene therapy: formulation and transfection properties, Gene Ther. 6 (1999) 1438–1447. [16] X. Tong, D.G. Engehausen, C.T. Freund, et al., The efficacy of adenovirusmediated gene therapy of ovarian cancer is enhanced by using the cytomegalovirus promoter, Anticancer Res. 18 (1998) 719–725. [17] T. Fukazawa, Y. Maeda, M.L. Durbin, et al., Pulmonary adenocarcinomatargeted gene therapy by a cancer- and tissue-specific promoter system, Mol. Cancer Ther. 6 (2007) 244–252. [18] I. Horikawa, P.L. Cable, S.J. Mazur, et al., Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression, Mol. Biol. Cell 13 (2002) 2585–2597. [19] T. Fukazawa, Y. Maeda, F.M. Sladek, et al., Development of a cancer-targeted tissue-specific promoter system, Cancer Res. 64 (2004) 363–369. [20] M.J. Brunda, L. Luistro, R.R. Warrier, et al., Antitumor and antimetastatic activity of interleukin 12 against murine tumors, J. Exp. Med. 178 (1993) 1223–1230.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model Yu Xu a,b, Zhengchun Liu b, Haiyan Kong b, Wenjie Sun a,b, Zhengkai Liao a,b, Fuxiang Zhou a,b, Conghua Xie a,b, Yunfeng Zhou a,b,⇑ a b
Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, PR China Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071, PR China
a r t i c l e
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Article history: Received 13 August 2011 Available online 22 August 2011 Keywords: Gene therapy hTERT promoter CArG element c-Irradiation HRP/IAA system Interleukin 12
a b s t r a c t The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Gene therapy is a novel approach for cancer treatment, which has been evaluated in a great deal of preclinical and clinical trials over the past years. However, side effects due to the lack of tumor specificity limited its application. Therefore, targeted gene therapy strategies were developed to prevent the toxicity of therapeutic gene in normal cells, cell- or tissue-specific promoter being the major one [1]. The human telomerase reverse transcriptase (hTERT) promoter which is highly activated in cancer cells but not in normal differentiated cells has been widely used to drive the expression of therapeutic genes in various cancer cells [2–4]. However, the transcriptional activity of hTERT promoter is weaker than the nonspecific cytomegalovirus (CMV) promoter that is commonly used, resulting in insufficient therapeutic gene expression [5]. Hence, additional regulatory strategies are needed to increase the transcriptional activity of the hTERT promoter.
⇑ Corresponding author at: Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, PR China. Fax: +86 27 67812889. E-mail addresses: [email protected] (Y. Xu), [email protected] (Z. Liu), [email protected] (H. Kong), [email protected] (W. Sun), fastbeta@ gmail.com (Z. Liao), [email protected] (F. Zhou), [email protected] (C. Xie), [email protected] (Y. Zhou). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.077
The early growth response-1 (Egr-1) promoter has been described as a radiation-inducible promoter, whose activation depends on CArG elements, a 10 nucleotide motifs of consensus sequence CC(A/T)6GG found in the promoter region of Egr-1 gene [6,7]. In order to reduce the nonspecific binding sites of the Egr-1 promoter, a novel chimeric promoter consisting of tandem repeats of isolated CArG elements and the CMV promoter was designed to drive suicide gene expression, which showed better radiation inducibility than the wild-type Egr-1 promoter [8,9]. In the study, we designed a novel dual radiation-inducible and tumor-specific promoter system by combining CArG elements and the hTERT promoter, which can target only to cancer cells as well as sufficient transcriptional activity. To test the potential clinical benefit of such an approach, a gene-directed enzyme prodrug therapy (GDEPT) system consisting of the horseradish peroxidase (HRP) gene driven by the chimeric promoter and the prodrug indole-3-acetic (IAA) was developed and its antitumor effect was evaluated in vitro and in vivo. Furthermore, cytokine-based immuno-gene therapy has been shown to be involved in the process of suicide gene therapy [10]. Interleukin 12 (IL12) is a heterodimeric cytokine that plays multiple roles in the immune system such as augmenting the proliferation and cytotoxic activity of T cells and NK cells [11]. Our previous study showed that the combination of HRP/IAA system and IL12 had cumulative antitumor effects [12]. Therefore, we also considered that adenovirus-mediated IL12 gene could enhance
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Fig. 1. Construction and characterization of synthetic promoter with tumor specificity and radiation inducibility. (A) Schematic drawing of the construction for the chimeric promoters. Luciferase gene was driven by the chimeric promoter consisting of repeated CArG elements (n = 4, 6, 8 or 10) and the hTERT promoter. (B) Tumor specificity of synthetic promoters. Luciferase reporter plasmids containing various promoters were introduced into LLC cells and MRC-5 cells which were telomerase- positive and negative, respectively. (C and D) Radiation inducibility of synthetic promoters. Transfected LLC cells were exposed at 4-Gy irradiation to screen for the optimal number of repeated CArG elements (C), and then treated with various doses (0, 2, 4, 6, 8, 10 Gy) of radiation to obtain the most inducible dose (D). The data of luciferase assay were expressed as relative activity with regard to pGL3-control, which was considered as 100%. Data represent means ± SD of at least three independent experiments.
the antitumor effect of HRP/IAA system driven by the novel chimeric promoter in a murine model of Lewis lung carcinoma (LLC). 2. Materials and methods 2.1. Cell culture LLC cell line and human MRC-5 cell line were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and maintained in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml streptomycin. Cells were incubated at 37 °C in an atmosphere containing 5% CO2. All culture reagents were purchased from Hyclone (Logan, UT, USA). 2.2. Animals Female C57BL/6 mice (6–8 weeks) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and housed in a specific pathogen-free (SPF) facility at the Animal Experimental Center of Wuhan University. The facilities and the protocol of this experiment were consistent with the regulations on animal use for biomedical experiments issued by the Ministry of Science and Technology of China and approved by the Animal Care Committee of Wuhan University.
phTERT-luc) as we described previously [13]. The synthetic enhancers containing different numbers of CArG elements were cloned as double-stranded linker molecules derived from complementary single-stranded oligonucleotides: 50 -CGCGTGATAT(CCTTA TTTGG)n-30 and 50 -AATT(CCAAATAAGG)nATATCA-30 (n = 4, 6, 8, 10). The synthetic enhancers were placed upstream of the hTERT promoter in phTERT-luc by using the MulI and EcoRI sites at the linker ends. This created four plasmids containing luciferase gene driven by chimeric promoters consisting of CArG elements and the hTERT promoter, which were pC4-hTERT-luc, pC6-hTERT-luc, pC8-hTERTluc and pC10-hTERT-luc, respectively. Schematic drawing of the construction for the chimeric promoters is available in Fig. 1A. 2.4. Luciferase assay The expression of the luciferase gene driven by the chimeric promoter was determined using the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instruction. Briefly, cells plated in 24-well culture plate at 80– 90% confluency were transfected with 0.4 lg of the luciferase reporter plasmid and 0.4 lg of the pRL-TK plasmid. Twenty four hours later, cells were irradiated using a 60Co gamma (c)-ray source (GWXJ80 type, Chengdu, China) and analyzed for radiation-induced gene expression by luciferase assay after another 24 h. The pRL-TK plasmid was used as an internal control. The simian virus 40 (SV40) promoter (pGL3-control) was used as a positive control and its luciferase activity was considered to be 100%.
2.3. Vector construction 2.5. Adenovirus construction The pGL3-basic plasmid (Promega, Madison, USA) was used as the basis for all the vector constructs. The hTERT promoter was placed upstream of the luciferase gene in pGL3-basic (creating
All adenovirus vectors were constructed using AdEasy System as we described previously [14]. Briefly, virus vectors expressing
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HRP gene driven by a chimeric promoter (AdC6-hTERT-HRP) and murine IL12 (mIL12) gene under the control of the CMV promoter (Ad-CMV-mIL12) were constructed by homologous recombination in Escherichia coli. Viruses were purified by double cesium chloride gradient ultracentrifugation. Viral tilter was determined by plaque assay and expressed as plaque forming unit (pfu). Purified virus aliquots were stored at 80 °C. 2.6. Western blotting LLC cells were infected with adenoviruses expressing HRP at multiplicity of infection (MOI) of 1000. This MOI was selected as previous experiments had demonstrated a maximal level of transduction efficiency using these experimental protocols [12]. Twenty four hours later, cells were irradiated and then incubated for another 24 h. HRP expression in treated LLC cells was determined by Western blotting. The mouse anti-HRP antibody (dilution, 1:500) and mouse anti-b-actin (dilution, 1:1000) were used to detect corresponding proteins. All the antibodies including goat antimouse secondary antibody (dilution, 1:10,000) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). 2.7. Cytotoxicity of HRP/IAA system in vitro Transduced LLC cells (2 103/well) were plated in 96-well culture plates and irradiated 24 h later, followed by IAA (Sigma, MO, USA) administration at a concentration of 0–5 mM. Media containing different concentration of IAA were exchanged every 48 h. Cell viability was determined using MTT assay (Invitrogen, CA, USA) 120 h later and optical density values were measured by a microplate reader (Turner BioSystems, CA, USA). 2.8. In vivo antitumor effects A total of 5 106 LLC cells were injected subcutaneously in the right flank of C57BL/6 mice. Tumors were isolated, prepared into cell suspension and inoculated into new mice 14 days later. When tumors reached 5 mm in diameter (day 10), the mice were randomized to treatment groups of 10 mice: Ad-CMV-GFP group, Ad-CMV-mIL12 group, AdC6-hTERT-HRP + irradiation (Trigger) group and combination group (AdCMVmIL-12 + AdC6-hTERTHRP + Trigger). The mice were treated with 1 109 pfu Ad-CMVGFP, 5 108 pfu Ad-CMV-mIL12 or 5 108 pfu AdCx-hTERT-HRP adenoviruses diluted in 30 ll PBS by intratumoral administration. Local c-irradiation (Trigger) was performed 24 h later (day 11). IAA (50 mg/kg daily) was administered to the mice by intraperitoneal injection for seven consecutive days from day 12 to 18. Twenty four hours later, five mice of each group were sacrificed for histological examination and toxicity study. The remaining mice were examined every 3 days for evaluation of tumor growth. Tumor sizes were measured using a caliper in mm3 and volumes were calculated using the following formula: (L W2)/2, where L equals length and W equals width. Study endpoint was death or sacrifice of the animal with very high tumor volume that exceeded 3500 mm3. 2.9. Immunohistochemistry Tumor tissues were fixed using formalin and 4 lm sections were stained with hematoxylin and eosin for routine histological analysis. For immunohistochemical analysis, fresh-frozen acetone-fixed sections were stained for infiltration T lymphocytes (CD4+ and CD8+) with specific antibodies (BD PharMingen, CA, USA) using standard peroxidase method. Positive staining was scored by light microscopy. After initial scanning under 100 magnification, positively stained cells in 10 fields under 400 magnifi-
Fig. 2. Expression (A) and biological activity (B) of exogenous HRP in vitro. LLC cells were infected with Ad-CMV-GFP and AdC6-hTERT-HRP with or without triggering by 6-Gy irradiation. (A) HRP expression in transduced LLC cells was detected by Western blotting. (B) To evaluate the biological activity of exogenous HRP, cytotoxicity was determined using MTT assay after IAA administration at indicated concentrations in transduced LLC cells. Data were expressed as percentage relative to drug-free cells. Each point represented the means ± SD of three independent experiments.
cation were counted and the mean number/high power field (HPF ± SEM) was determined. 2.10. In vivo toxicity studies Biochemistry parameters including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in the sera from treated animals were measured to evaluate side effects of treatment. Key organs and tissues were harvested, fixed with formalin and stained with hematoxylin and eosin for histological analysis. Three mice without any treatment were used as normal control. 2.11. Statistical analysis The significance of differences between experimental groups was calculated using Student’s t-test or one-way ANOVA analysis as appropriate. Statistical analyses were performed with SPSS software version 13.0 (SPSS Inc. Chicago, IL) and two-tailed P < 0.05 was considered as statistically significant.
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3. Results 3.1. Characterization of synthetic promoters To evaluate the specificity of chimeric promoters, LLC cells and MRC-5 cells were transfected with various plasmids constructed. As shown in Fig. 1B, the hTERT promoter and synthetic promoters showed higher activity in LLC cells than in MRC-5 cells (P < 0.01). Then transfected LLC cells were treated with 4-Gy c-irradiation to evaluate the inducibility of chimeric promoters (Fig. 1C). The results showed that the C6-hTERT promoter was more inducible than other promoters (C6-hTERT vs hTERT, 1.38-folds, P = 0.000; C6hTERT vs C4-hTERT, 1.19-folds, P = 0.000; C6-hTERT vs C8-hTERT, 1.18-folds, P = 0.001; C6-hTERT vs C10-hTERT, 1.28-folds, P = 0.000). LCC cells were transfected with pC6-hTERT-luc and treated with various doses (0, 2, 4, 6, 8, 10 Gy) of c-irradiation (Fig. 1D). It was evident that the level of luciferase expression observed was dependent on radiation dose, with maximal expression seen after 6 Gy (6 vs 0 Gy, 1.61-folds, P = 0.000; 6 vs 2 Gy, 1.45folds, P = 0.001; 6 vs 4 Gy, 1.2-folds, P = 0.030; 6 vs 8 Gy, 1.23-folds, P = 0.017; 6 vs 10 Gy, 1.45-folds, P = 0.001). Therefore, the pC6hTERT promoter and 6-Gy irradiation were chosen for the subsequent studies. 3.2. HRP expression in LLC cells in vitro
tumor growth was more inhibited in the combination group than treatment with Ad-hTERT-HRP alone without radiation (p = 0.001 on days 12–36) [12]. As assessed by microscopic examination, the therapeutic effects of combination strategy were more significant; the tumor nodules in combination groups contained larger necrotic areas than other groups (data not shown). The results indicate the potent potentials of the combination strategy for cancer treatment. 3.5. T lymphocytes infiltration in tumors To explore the mechanism of cumulative antitumor effects, the number and distribution of CD4+ and CD8+ T cells in tumor tissues were evaluated using IHC. As shown in Fig. 4A–H, both CD4 and CD8 were stained in the cell membrane of interstitial infiltrates. The combination group showed extensive infiltration of CD4+ T cells and CD8+ T cells in comparison with Ad-CMV-GFP group (CD4, 8.6-folds, P = 0.000; CD8, 8.1-folds, P = 0.000), Ad-CMVmIL12 group (CD4, 1.4-folds, P = 0.003; CD8, 1.5-folds, P = 0.000) and AdC6-hTERT-HRP (CD4, 1.9-folds, P = 0.000; CD8, 2.8-folds, P = 0.000) (Fig. 4I). 3.6. Toxicity studies in vivo To evaluate the toxicity of various strategies in vivo, biochemistry parameters of liver and kidney in sera and histological changes
LLC cells infected by Ad-CMV-GFP or AdC6-hTERT-HRP with or without 6-Gy irradiation treatment were harvested for determining HRP expression by Western blotting. The results showed that the HRP expression was observed in AdC6-hTERT-HRP transduced groups whereas not in Ad-CMV-GFP transduced groups (Fig. 2A). In addition, a significant increase in the HRP expression was observed after irradiation (1.69-folds, P = 0.000). These data suggest that the therapeutic genes were successfully delivered into the cells and the novel chimeric promoter resulted in increased transcriptional activity. 3.3. Cytotoxicity of the HRP/IAA system in vitro Transduced LLC cells were treated with IAA at indicated concentrations in Fig. 2B. The data were presented as percentages of cell viability with regard to control cells without IAA treatment. LLC cells in the AdC6-hTERT-HRP group and AdC6-hTERT-HRP + Trigger group exhibited a dose-dependent cytotoxicity, and the IC50 of IAA was about 3.0 and 1.5 mM, respectively. The results indicate that the HRP/IAA system controlled by chimeric promoter could be induced by radiation, which had sufficient cytotoxic effects on LLC cells. 3.4. Suppression of tumor growth in vivo To further evaluate the antitumor activity of AdC6-hTERT-HRP/ IAA and the cumulative antitumor effect of combining AdC6hTERT-HRP/IAA and Ad-CMV-mIL12 in vivo, we established an immunocompetent tumor model with LLC cells in C57BL/6 mice. The mice were assigned randomly to four groups (group 1, AdCMV-GFP; group 2, Ad-CMV-mIL12; group 3, AdC6-hTERTHRP + Trigger; group 4, Ad-CMV-mIL12 + AdC6-hTERT-HRP + Trigger). After IAA administration, the expression of HRP and mIL12 in the tumor tissues was detected by Western blot as shown in Fig. 3A. The tumor growth was monitored every 3 days (Fig. 3B). The data showed that the combination treatment with Ad-CMVmIL12 and AdC6-hTERT-HRP + Trigger resulted in remarkable inhibition of tumor growth in comparison with the other three groups (groups 4 vs 1, p = 0.034 on days 6–24; groups 4 vs 2, p = 0.046 on days 9–36; groups 4 vs 3, p = 0.000 on days 12–45). Moreover, the
Fig. 3. Tumor suppression in LLC mouse model. Subcutaneous LLC tumors were treated by intratumoral injection with Ad-CMV-GFP, Ad-CMV-mIL12 and AdC6hTERT-HRP + Trigger alone or in combination. (A) Expression of HRP and mIL12 in the tumor tissues were detected by Western blot. This result is representative of that obtained from three independent experiments. (B) Tumor volumes were monitored at the indicated time points after IAA administration. Each data point represented the mean tumor volume in that group. Error bars represented means ± SD.
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Fig. 4. Infiltration of CD4+ and CD8+ T lymphocytes in treated LLC tumors. Five treated mice of each group were sacrificed 24 h after last IAA administration. Tumor sections were analyzed by immunohistochemical staining using specific antibodies against CD4 (A–D) or CD8 (E–H). The positive cells were scored through light microscopy (I). After initial scanning under 100 magnification, positive stained cells in 10 filed under 400 magnification were counted and the mean number of stained cells was averaged over 10 fields.
of key tissues were examined for treated mice. The results of liver and kidney function tests of all treated animals were not statistically different from those of control animals (Table 1). Meanwhile, there were no marked pathological changes in heart, liver, spleen, lung and kidney of treated mice (data not shown). These data suggest that the treatment was well tolerated. 4. Discussion Gene therapy is among the approaches currently used to treat malignant tumors, which uses specific therapeutic genes that cause death in cancer cells. The adenoviral vector has been used as a transfer vehicle to introduce genes into tumor cells since it is more efficient than non-viral gene transfer methods [15]. In early attempts of gene therapy, therapeutic genes were driven by ubiquitous promoters such as the CMV promoter, which induce nonspecific toxicity to normal cells and tissues in addition to cancer cells [16]. Subsequently, novel tumor- and tissue-specific promoter systems have been developed to target tumor cells but not normal cells for reducing undesirable toxicity [17]. The promoter region of tumor specific hTERT was cloned and characterized, which was used to induce tumor-specific therapeutic gene expression in cancer gene therapy [18,19]. However, gene expression driven by the hTERT promoter is much lower than expression driven by commonly used viral promoters, such as CMV promoter and
Table 1 Serum biomarkers of liver and kidney function in each group (means ± SD). Group
ALT (U/L)
AST (U/L)
BUN (mg/dl)
Cr (lmol/L)
Normal Ad-CMV-GFP Ad-CMV-mIL12 AdC6-hTERT-HRP Combination F value P*
42.7 ± 2.70 45.3 ± 2.61 43.8 ± 2.38 44.7 ± 1.96 45.2 ± 1.73 0.350 0.841
124.9 ± 13.40 128.8 ± 9.91 130.4 ± 13.79 124.9 ± 9.68 129.8 ± 7.51 0.266 0.896
12.7 ± 2.00 13.8 ± 0.89 13.6 ± 0.82 12.7 ± 1.40 12.8 ± 1.43 0.806 0.537
6.7 ± 1.10 6.8 ± 0.47 7.2 ± 0.43 6.7 ± 0.74 7.1 ± 0.29 0.655 0.631
It was shown that there were no statistically significant differences in serum ALT, AST, BUN and Cr levels among different groups (P > 0.05). ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine. * P < 0.05 was considered to be statistically significant.
simian virus 40 (SV40) promoter [5]. It is necessary, then, to increase the activity of this promoter. In the current study, an attempt was made to maintain the tumor specificity of the hTERT promoter and to increase its activity by using a radiation-inducible CArG element. We constructed several synthetic hTERT promoters containing different number of CArG elements, which were introduced into hTERT positive (LLC) and negative (MRC-5) cells to show the difference in transcriptional activity between tumor and normal cells. The chimeric pro-
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moters showed higher transcriptional activity in LLC cells than in MRC-5 cells, suggesting the tumor specificity (Fig. 1B). Then the synthetic promoter with six repeated CArG elements was found to be more radiation-inducible than the others (Fig. 1C). Further study was performed to obtain the optimal dose that resulted in more induction, and the peak activity was found in cells treated with 6-Gy irradiation (Fig. 1D). Taking these data together, the C6-hTERT promoter triggered by 6-Gy irradiation showed the stronger transcriptional activity besides tumor specificity. Adenovirus vector containing the HRP gene under control of the C6-hTERT promoter was constructed and introduced into LLC cells. Using Western blot analysis (Fig. 2A), we detected HRP in AdC6hTERT-HRP infected LLC cells and the expression level was higher after triggering with 6-Gy irradiation, suggesting that therapeutic gene was successfully delivered by adenovirus vectors and the novel chimeric hTERT promoter increased the target gene expression. We further investigated the in vitro effects of HRP expression following IAA treatment and observed an IAA dose-dependent decrease in cell viability in LLC cells, which indicated the biological activity of exogenous HRP (Fig. 2B). Additionally, the AdC6hTERT-HRP + Trigger group showed the strongest cytotoxic effect at low concentration of IAA, suggesting that the increased HRP expression by the radiation via chimeric hTERT promoter could strengthen the killing effect of the HRP/IAA system. Delivery of cytokine genes to cancerous cells as cancer gene immunotherapy alters the local tumor environment and induces an anti-tumor immune response to facilitate tumor eradication [20]. We previously showed that the combination of HRP/IAA system and IL12 had cumulative antitumor effects [12]. In the study, we hypothesized that combining HRP/IAA system controlled by radiation-inducible and tumor-specific C6-hTERT promoter and IL-12 gene driven by the CMV promoter could produce more significant antitumor effects. To validate this hypothesis, we established an immunocompetent LLC murine model. As expected, the combination strategy showed stronger antitumor effects in comparison with single agent strategies (Fig. 3B). There are two possible explanations for the cumulative antitumor effects: increased infiltration of stimulated helper CD4+ T cells and cytolytic CD8+ T cells induced by the local expression of IL12 and further activation of the host antitumor immune response mediated by the enhanced cell killing effect of the HRP/IAA system driven by the chimeric promoter (Fig. 4). Although the recruitment of CD4+ and CD8+ T cells was lower, the triggered AdC6-hTERT-HRP vector inhibited tumor growth more than the Ad-CMV-mIL12 vector, which might be because the killing effects of the suicide gene system (including direct killing effect and by stander effect) were more directly and stronger than the immune-gene. Moreover, there were no pathological changes in the serum biomarkers of liver and kidney function (Table 1) and the histological examination of key organs, suggesting that the treatment was well tolerated. In this study, we designed a novel chimeric promoter which maintained the tumor specificity by hTERT promoter and increased the expression of therapeutic gene by the radiation-inducible element. The combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.
Acknowledgments We thank Dr. Lin Huang from Department of Pathology, University of Nebraska Medical Center and Dr. Jinxuan Hou from Department of Oncology, Zhongnan Hospital of Wuhan University to review the manuscript. This study was supported by Grants from the National Natural Science Foundation of China (No. 30672438), the Hubei Provincial Natural Science Foundation of China (Nos. 2006ABC009 and JX4A06) and the Scientific and Technological Project of Wuhan City (No. 200960323129). References [1] C. Wu, J. Lin, M. Hong, et al., Combinatorial control of suicide gene expression by tissue-specific promoter and microRNA regulation for cancer therapy, Mol. Ther. 17 (2009) 2058–2066. [2] J. Gu, S. Kagawa, M. Takakura, et al., Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers, Cancer Res. 60 (2000) 5359– 5364. [3] A.S. Majumdar, D.E. Hughes, S.P. Lichtsteiner, et al., The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters, Gene Ther. 8 (2001) 568–578. [4] D. Jacob, M. Bahra, G. Schumacher, et al., Gene therapy in colon cancer cells with a fiber-modified adenovector expressing the TRAIL gene driven by the hTERT promoter, Anticancer Res. 24 (2004) 3075–3079. [5] J.S. Song, Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer, Biosci. Biotechnol. Biochem. 68 (2004) 1634–1639. [6] M.A. Stackhouse, D.J. Buchsbaum, Radiation to control gene expression, Gene Ther. 7 (2000) 1085–1086. [7] R. Datta, E. Rubin, V. Sukhatme, et al., Ionizing radiation activates transcription of the EGR1 gene via CArG elements, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 10149–10153. [8] B. Marples, S.D. Scott, J.H. Hendry, et al., Development of synthetic promoters for radiation-mediated gene therapy, Gene Ther. 7 (2000) 511–517. [9] S.D. Scott, M.C. Joiner, B. Marples, Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy, Gene Ther. 9 (2002) 1396–1402. [10] S. Kuriyama, H. Tsujinoue, H. Yoshiji, Immune response to suicide gene therapy, Methods Mol. Med. 90 (2004) 353–369. [11] M.P. Colombo, G. Trinchieri, Interleukin-12 in anti-tumor immunity and immunotherapy, Cytokine Growth Factor Rev. 13 (2002) 155–168. [12] Y. Xu, J. Hou, Z. Liu, et al., Gene therapy with tumor-specific promoter mediated suicide gene plus IL-12 gene enhanced tumor inhibition and prolonged host survival in a murine model of Lewis lung carcinoma, J. Transl. Med. 9 (2011) 39. [13] Z.K. Liao, F.X. Zhou, Z.G. Luo, et al., Radio-activation of hTERT promoter in larynx squamous carcinoma cells: an ‘indirected-activator’ strategy in radiogene-therapy, Oncol. Rep. 19 (2008) 281–286. [14] J. Luo, Z.L. Deng, X. Luo, et al., A protocol for rapid generation of recombinant adenoviruses using the AdEasy system, Nat. Protoc. 2 (2007) 1236–1247. [15] Y.P. Zhang, L. Sekirov, E.G. Saravolac, et al., Stabilized plasmid–lipid particles for regional gene therapy: formulation and transfection properties, Gene Ther. 6 (1999) 1438–1447. [16] X. Tong, D.G. Engehausen, C.T. Freund, et al., The efficacy of adenovirusmediated gene therapy of ovarian cancer is enhanced by using the cytomegalovirus promoter, Anticancer Res. 18 (1998) 719–725. [17] T. Fukazawa, Y. Maeda, M.L. Durbin, et al., Pulmonary adenocarcinomatargeted gene therapy by a cancer- and tissue-specific promoter system, Mol. Cancer Ther. 6 (2007) 244–252. [18] I. Horikawa, P.L. Cable, S.J. Mazur, et al., Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression, Mol. Biol. Cell 13 (2002) 2585–2597. [19] T. Fukazawa, Y. Maeda, F.M. Sladek, et al., Development of a cancer-targeted tissue-specific promoter system, Cancer Res. 64 (2004) 363–369. [20] M.J. Brunda, L. Luistro, R.R. Warrier, et al., Antitumor and antimetastatic activity of interleukin 12 against murine tumors, J. Exp. Med. 178 (1993) 1223–1230.