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Molecular Cancer Therapeutics 6, 244-252, January 1, 2007. doi: 10.1158/1535-7163.MCT-06-0408
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

Pulmonary adenocarcinoma–targeted gene therapy by a cancer- and tissue-specific promoter system

Takuya Fukazawa1, Yutaka Maeda2, Mary L. Durbin3, Toru Nakai1, Junji Matsuoka1, Hirotoshi Tanaka4, Yoshio Naomoto1 and Noriaki Tanaka1

1 First Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; 2 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; 3 Department of Ecology and Evolutionary Biology, University of California, Irvine, California; and 4 Division of Clinical Immunology and Department of Rheumatology and Allergy, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Requests for reprints: Takuya Fukazawa, First Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Phone: 81-86-235-7257; Fax: 81-86-221-8775. E-mail: FukazawaT{at}aol.com

Abstract

Gene therapy is one of the approaches used to treat lung cancer. The benefit of cancer gene therapy is that different types of tumors can be selectively targeted by tumor-specific expression of therapeutic genes that include an apoptosis gene to destroy the tumor. Previously, we described a promoter (TTS promoter) that we designed that is specifically targeted to lung cancer cells but not to other types of cancer or normal cells including stem cells. In this pursuit, we further characterize the specificity of the TTS promoter in four types of lung cancer cells (squamous cell lung carcinoma, pulmonary adenocarcinoma, small-cell lung carcinoma, large-cell lung carcinoma). The TTS promoter is highly active only in pulmonary adenocarcinoma cells but not in the other three types of lung cancer cells. The specificity seems to be derived from transcription factor thyroid transcription factor 1–associating cofactors that affect human surfactant protein A1 promoter activity in pulmonary adenocarcinoma. We inserted the proapoptotic gene Bcl-2–associated X protein (Bax) into the TTS promoter (TTS/Bax). The TTS/Bax selectively causes BAX expression and cell death in pulmonary adenocarcinoma but not in other cells. Cell death caused by the BAX expression was also observed in pulmonary adenocarcinoma that is resistant to the anticancer drug gefitinib (epidermal growth factor receptor tyrosine kinase inhibitor). BAX expression and cell death can be suppressed by dexamethasone (a glucocorticoid) treatment through negative glucocorticoid elements in the TTS promoter. Here we report a drug-controllable TTS/Bax system targeting pulmonary adenocarcinoma. [Mol Cancer Ther 2007;6(1):244–52]

Introduction

Cancer gene therapy using the transfer of the proapoptotic suicide gene into cancer cells has already been attempted to treat tumors (1, 2). Several cancer-specific promoters, including the hTERT (a telomerase component) promoter, were developed to target cancer cells, but their promoter activities are also high in many kinds of stem cells and cirrhotic liver (3–8). Therefore, the use of these cancer-specific promoter–mediated gene expression systems might cause unknown side effects in these cells and tissues. Thyroid transcription factor 1 (TTF1) and human surfactant protein A1 (hSPA1) are expressed in normal human lung respiratory epithelium (9, 10). TTF1 binds and transactivates lung-specific promoters including hSPA1 promoter (11, 12). By combining the cancer specificity of the hTERT promoter and the lung specificity of TTF1-hSPA1 promoter, a dual system called the TTS (TTF1 gene under the control of hTERT promoter and hSPA1 promoter) system was developed to target lung cancer cells (13). The TTS system contains negative glucocorticoid elements from the hSPA1 promoter (14) so that the TTS system can be controlled by dexamethasone (13).

To test which types of lung cancer cells are targeted by the TTS system, we have investigated the applicability of the TTS system in four types of lung cancer (squamous cell lung carcinoma, pulmonary adenocarcinoma, small-cell lung carcinoma, and large-cell lung carcinoma) and mesothelioma cells. We tested the TTS system in breast cancer cells, liver cancer cells, normal lung fibroblasts, and stem cells to assess the tissue and cancer specificity of the TTS system. The proapoptotic gene Bcl-2–associated X protein (Bax) inserted into the TTS system (TTS/Bax) selectively killed pulmonary adenocarcinoma. The TTS/Bax system did not affect normal cells, including stem cells. The BAX expression by the TTS promoter was suppressed by dexamethasone.

The recently developed anticancer drug gefitinib that targets epidermal growth factor receptor (EGFR) has been expected to be a better drug to treat non–small-cell lung cancer patients than chemotherapeutic drugs previously used. However, gefitinib targets only tumors containing somatic mutations of EGFR but not the wild-type EGFR (15). The incidence of EGFR mutations is higher in East Asia than in the United States and Europe (16). Moreover, pulmonary adenocarcinoma carrying the K-ras mutation G12C is resistant to gefitinib (17–19). This mutation happens more frequently in the United States and Europe than in East Asia (20). In this study, TTS/Bax induced cell death in the gefitinib-resistant pulmonary adenocarcinoma A549, H441, and H358 cells that have wild-type EGFR and K-ras codon 12 mutation. To provide gene therapy that targets only lung cancer cells but not normal tissues, we have developed a tissue- and cancer-specific promoter system (13). Our data support the potential usefulness of a novel and safe gene therapy using a tissue- and cancer-specific promoter system targeting pulmonary adenocarcinoma.

Materials and Methods

Plasmids and Recombinant Adenovirus Vectors
Construction of the recombinant adenovirus vectors Ad-Bicistronic hTERT/TTF1·hSPA1-157 5x/GFP (Ad-TTS/GFP) and Ad-hTERT/GFP was as previously described (13). The hemagglutinin-tagged human Bax (HA-Bax) gene was kindly provided by Dr. Korsemyer (Departments of Pathology and Medicine, Harvard Medical School, Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Boston, MA; ref. 21). The plasmids phTERT/Bax and the phTERT/TTF1·hSPA1-157 5x/Bax were constructed by excising HA-Bax fragment from pSFFV-LTR neo/HA-Bax and ligating it into pGL3.hTERT Luc or pGL3.hSPA1-157 5x/ Luc (13) after excising luciferase gene. The recombinant adenovirus vector Ad-Bicistronic hTERT/TTF1·hSPA1-157 5x/Bax (Ad-TTS/Bax) was generated by homologous recombination (22) and was plaque purified. The optimal multiplicity of infection was determined by infecting each cell line with Ad-CMV/GFP and assessing the expression of green fluorescent protein (GFP) by flow cytometric analysis. The H69 and H146 human small-cell lung cancer cells were infected with the recombinant adenoviruses at a multiplicity of infection of 4,000 viral particles per cell and all other human cells were infected at a multiplicity of infection of 2,000 viral particles per cell.

Cell Lines and Culture Conditions
Human large-cell lung carcinoma cells H460, H661, and H1915; human small-cell carcinoma cells H69 and H146; human lung adenocarcinoma cells A549, H441, H358, and H322; human squamous cell lung carcinoma cells H226; human breast cancer cells MCF7; and human mesothelioma cells MSTO-211H and H2542 were obtained from the American Type Culture Collection (Manassas, VA) and grown in Ham's F12 (A549 cells), RPMI 1640 (H460, H661, H1915, H69, H146, H358, H322, and H226 cells) with high-glucose Dulbecco's modified Eagle medium (H441 and MCF7 cells) supplemented with 10% heat-inactivated fetal bovine serum. Human squamous cell lung carcinoma cells SQ5, LK2, EBC1, and EBC2 and human small-cell carcinoma cells SBC3, SBC4, SBC5, and SBC9B were kindly provided by Dr. Kiura Katsuyuki (Second Department of Internal medicine, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; refs. 23–26) and grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. Human hepatoblastoma cells Hep3B were maintained in Eagle's minimal essential medium supplemented with 1 mmol/L sodium pyruvate, 0.1% nonessential amino acids, and 10% heat-inactivated fetal bovine serum. Normal human lung fibroblast cells NHLF and human mesenchymal stem cells obtained from Clonetics (San Diego, CA) and mouse embryonic stem cells obtained from Specialty Media (Phillipsburg, NJ) were grown in culture medium supplied by the manufacturer. All cell lines were cultured in 10% CO2 at 37°C.

RNA Extraction and Reverse Transcription-PCR Analysis
Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform method followed by DNase treatment. One entire plate of cells in a six-well plate was used for isolation of total RNA. For cDNA synthesis, oligo dT primers were used to prime the reverse transcription reactions. The primer sequences were, for TTF1, 5'-ccagcatgatccacctgac-3' and 5'-actgctgctgagcctgttg-3', and, for hSPA1, 5'-ctgtcccaaggaatccagag-3' and 5'-ttccactgcccatctgtgta-3'. The primer sequences for glyceraldehyde-3-phosphate dehydrogenase were 5'-cagccgagccacatc-3' and 5'-tgaggctgttgtcatacttct-3'. The cycling conditions were as follows: 95°C for 1 min, 63°C for 1.5 min, 72°C for 2 min for 30 cycles for TTF1; 95°C for 1 min, 58°C for 1.5 min, 72°C for 2 min for 30 cycles for hSPA1 and glyceraldehyde-3-phosphate dehydrogenase. The amplified PCR products were electrophoretically separated on 1% agarose gel and visualized by ethidium bromide staining. The expected sizes of PCR products were 177 bp for TTF1, 354 bp for hSPA1, and 459 bp for glyceraldehyde-3-phosphate dehydrogenase.

Immunohistochemistry of TTF1 and hSPA1
Forty-eight hours after plating in chamber slides, the cells were washed once with PBS. The primary antibodies of prediluted TTF1 (clone 8G7G3/1) and hSPA1 (clone PE-10; DAKO, San Diego, CA) were used at 1:100 dilution for TTF1 and at 1:50 dilution for hSPA1 with no pretreatment. Cells were then stained by the avidin-biotin peroxidase complex method according to the procedure provided by the manufacturer (DAKO). With respect to immunostaining, the cells were classified into a positive group and a negative group with <1% positive cells.

Transient Transfection Reporter Assays
All transfections were carried out in six-well plates. Cells were seeded 36 h before transfection at the following densities: 0.50 x 106 per well for NHLF cells and 0.40 x 106 per well for all other cells. Transfection for the human small-cell lung carcinoma cells H69 and H146 was carried out with Fugene 6 (Roche, Basel, Switzerland) and that for all other cells was done with Lipofectin (Invitrogen life Technologies, Carlsbad, CA) in accordance with the manufacturers' protocols. All the transfected cells were harvested at 24 h. Results of one representative experiment are presented as fold induction of relative light units normalized to ß-galactosidase activity relative to that observed for the control vectors. Each experiment was repeated at least thrice. Error bars indicate the SD from the average of triplicate samples in one experiment.

Immunoblot Analysis
Cells in six-well plates were washed once with ice-cold PBS containing 5 mmol/L EDTA and 1 mmol/L sodium orthovanadate and harvested by scraping into 0.2 mL/well ice-cold lysis buffer [1% Triton X-100, 20 mmol/L Tris-HCl (pH 8.0), 137 mmol/L NaCl, 10% glycerol (v/v), 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate (v/v)]. Cell lysates were clarified by centrifugation (10 min at 16,000 x g at 4°C) and protein concentration was determined using the detergent-compatible protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were electrophoretically transferred to a Hybond polyvinylidene difluoride transfer membrane (Amersham, Arlington Heights, IL). The membrane was incubated with primary and secondary antibodies according to the SuperSignal West Pico chemiluminescence protocol (Pierce, Rockford, IL) to detect secondary antibody binding. Antibody against the human TTF1 (clone 8G7G3/1) was purchased from DAKO. Antiactin and antihemagglutinin antibodies were obtained from Sigma (St. Louis, MO). Secondary horseradish peroxidase–conjugated goat anti-rabbit and anti-mouse antibodies were obtained from Jackson ImmunoRresearch Laboratories (West Grove, PA).

Flow Cytometric Analysis for Apoptosis
Cells were plated in 24-well plates at a density of 2 x 105 per well 1 day before the recombinant adenoviral vectors infection. After 72-h infection, both adherent cells and floating cells were harvested and washed once with PBS. Cells were resuspended in PBS containing 0.2% Triton X-100 and 1 mg/mL RNase for 5 min at room temperature and then stained with propidium iodide at 50 µg/mL to determine subdiploid DNA content using a FACScan. Doublets, cell debris, and fixation artifacts were gated out and sub-G0-G1 DNA content was determined using Cell Quest version 3.3 software.

Nucleofection
Cells (2 x 106) were used in each transfection experiment with the Nucleofector (Amaxa Biosystems GmbH, Cologne, Germany). Cells were nucleofected in an electroporation cuvette along with nucleofector solution and 4 µg of pCMV/Bax, phTERT/Bax, or pGL3.hSPA1-157 5x/Bax (pTTS/Bax) using the program A-23 for mesenchymal stem cells and A-30 for embryonic stem cells. Once nucleofected, cells were transferred into fresh media with the serum.

Measurement of Caspase-3 Activity
Caspase-3 activity was determined using the CaspACE Assay System, Colorimetric (Promega, Madison, WI) according to the manufacturer's protocol. Fold increase in protease activity was determined by comparing the level of caspase activity in treated cells with that in untreated cells.

Hoechst Staining
The morphologic characteristics of apoptosis were evaluated using Hoechst 33342 dye (Molecular Probes, Eugene, OR), which stains the DNA of cells. Cells were incubated with 1 µg/mL Hoechst dye and then visualized under a Zeiss-Jenalumar fluorescence microscope.

Results

Analysis of TTF1 and hSPA1 mRNA and Protein Expression in Cancer Cell Lines
The TTS system uses the transcription factor TTF1 and the human surfactant protein promoter A1 (hSPA1 promoter) to make the system lung specific (Fig. 1 ). To identify the types of lung cancer that can be targeted by the TTS system, we investigated the levels of TTF1 and hSPA1 mRNA and protein in 24 cell lines including four types of lung cancer (squamous cell carcinoma, adenocarcinoma, small-cell carcinoma, and large-cell carcinoma), mesothelioma, hepatoblastoma, breast cancer, and normal lung fibroblast. TTF1 mRNA was detected in lung squamous cell carcinoma cells (H226), pulmonary adenocarcinoma cells (H441, H358, PC3, and H322), small-cell lung carcinoma cells (SBC4 and SBC9B), and large-cell carcinoma cells (H661). TTF1 protein was expressed in lung squamous cell carcinoma cells (H226), pulmonary adenocarcinoma cells (H441, H358, and PC3), and large-cell carcinoma cells (H661; Fig. 2 ). The difference in expression between TTF1 mRNA and protein could be derived from the TTF1 protein stability depending on cell types. The hSPA1 mRNA was detected in pulmonary adenocarcinoma (H441, H358, PC3, and H322) and lung squamous cell carcinoma (H226) whereas the hSPA1 protein was detected only in pulmonary adenocarcinoma (H441 and PC3; Fig. 2). Again, the difference in the expression between hSPA1 mRNA and protein could be derived from the hSPA1 protein stability depending on cell types. Our results indicate that TTF1 is required but is not sufficient for hSPA1 protein expression. Together with previous work (27), these results suggest the presence of transcriptional/translational cofactors that exist in pulmonary adenocarcinoma.


Figure 1
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  		Figure 1. Schematic representation of the TTS system.

 

Figure 2
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  		Figure 2. Analysis of TTF1 and hSPA1 expression by reverse transcription-PCR and immunohistochemistry. A, detection of TTF1 and hSPA1 mRNA by reverse transcription-PCR in various kinds of cells. The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a control. Lanes: 1, SQ5; 2, LK2; 3, EBC1; 4, EBC2; 5, H226; 6, H441; 7, H358; 8, PC3; 9, H322; 10, A549; 11, H69; 12, H146; 13, SBC3; 14, SBC4; 15, SBC5; 16, SBC9B; 17, H460; 18, H661; 20, 211H; 21, H2542; 22, MCF7; 23, Hep3B; 24, NHLF. B, immunohistochemistry of TTF1 and hSPA1. Immunohistochemical positivity with TTF1 antibody (clone 8G7G3/1) was detected in the nucleus of pulmonary adenocarcinoma cells PC3 and large-cell lung carcinoma cells H661. The positivity with hSPA1 antibody (clone PE10) was detected in the cytoplasm of pulmonary adenocarcinoma cells PC3. No positivity with TTF1 or hSPA1 antibody was detected in squamous cell lung carcinoma SQ5 cells and pulmonary adenocarcinoma A549 cells. C, summary of TTF1 and hSPA1 expressions. Results of immunohistochemistry were classified into a positive group (+) and a negative group (–) with <1% positive cells.

 
TTS Generated Higher Transcriptional Activity in Human Pulmonary Adenocarcinoma Cells than in Other Types of Cells
To test the tissue and cancer specificity of the TTS system, we compared the TTS promoter activity with the hTERT (a telomerase component) promoter activity in four types of lung cancer cells (squamous cell lung carcinoma, pulmonary adenocarcinoma, small-cell lung carcinoma, and large-cell lung carcinoma), mesothelioma cells, hepatoblastoma cells, breast cancer cells, and normal lung fibroblasts. As shown in Fig. 3A , hTERT promoter activity was higher in all human cancer cells (6.38- to 25.90-fold) than in normal human lung cells (NHLF; 1.89-fold). These results are consistent with previous studies (28–30). On the other hand, the TTS promoter generated significantly higher activity (13.48- to 49.50-fold) in the human pulmonary adenocarcinoma cells (H441, H358, PC3, H322, and A549) than in other cells (Fig. 3A). Interestingly, exogenously expressed TTF1 driven by the hTERT promoter was sufficient to induce the TTS promoter activity even in the TTF1-negative pulmonary adenocarcinoma H322 and A549 cells. Moreover, overexpression of TTF1 by adenovirus vector Ad-TTS/GFP infection was also enough to induce endogenous hSPA1 mRNA in A549 cells but not in squamous cell carcinoma cells (SQ5) or large-cell carcinoma cells (H661; Fig. 3B). These results suggest the presence of pulmonary adenocarcinoma–specific transcriptional cofactors that affect the TTS promoter.


Figure 3
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  		Figure 3. Transcriptional activity of hTERT and TTS promoter in various kinds of cells. A, transient transfection reporter assays in 19 lung cancer cells, other types of cancer cells (Hep3B and MCF7), pleural mesothelioma cells (211H and H2542), and normal lung fibroblasts (NHLF) with pGL3.Basic (2 µg), pGL3.hTERT/Luc (2 µg), or pGL3.TTS/Luc (2 µg), plus pCMV.ß-gal (2 µg). Columns, fold induction of relative light units normalized to ß-galactosidase activity relative to that observed for control constructs. Triplicate experiments were done for each cell. Ca, carcinoma; Meso, mesothelioma. B, adenoviral-mediated transfer of TTF1 up-regulated hSPA1 mRNA in A549 pulmonary adenocarcinoma cells. Reverse transcription-PCR analysis of hSPA1 mRNA in Ad-CMV/GFP– or Ad-TTS/GFP–infected cells. Total RNA was prepared 24 h after adenoviral infection. The expression level of glyceraldehyde-3-phosphate dehydrogenase is shown as a control.

 
Ad-TTS/Bax Induced BAX Expression and Cell Death in Pulmonary Adenocarcinoma
To induce cell death in pulmonary adenocarcinoma by the TTS system, the proapoptotic Bax gene was inserted into the TTS system in place of GFP (Ad-TTS/Bax; Fig. 4A ). As shown in Fig. 4B, TTF1 was expressed in three different lung cancer cell types (squamous cell carcinoma SQ5 cells, pulmonary adenocarcinoma PC3 and H441 cells, and large-cell carcinoma H661 cells) by Ad-TTS/Bax due to the cancer-specific hTERT promoter activity. On the other hand, exogenous BAX (HA-BAX) was expressed only in pulmonary adenocarcinoma cells PC3 and H441 but there was little expression in lung squamous cell carcinoma SQ5 cells and lung large-cell carcinoma H661 cells. The cell death caused by Ad-TTS/Bax was examined by sub-G0-G1 DNA content using propidium iodide staining and flow cytometry 72 h after Ad-TTS/Bax infection. As shown in Fig. 4C, infection with the Ad-TTS/GFP control caused little DNA fragmentation (range, 0.67–4.51%) in all kinds of cells indicated (top). On the other hand, Ad-TTS/Bax infection caused a marked increase in sub-G1 DNA content only in lung adenocarcinoma PC3 cells (42.16%) but not in squamous cell carcinoma SQ5 cells (1.84%), small-cell carcinoma H146 cells (1.26%), large-cell carcinoma H661 cells (4.7%), hepatoblastoma Hep3B cells (5.07%), or normal lung fibroblast NHLF cells (3.98%; bottom). These results are consistent with the BAX expression in Fig. 4B. Pulmonary adenocarcinoma A549, H441, and H358 cells are known as EGFR tyrosine kinase inhibitor (gefitinib)–resistant cells that carry wild-type EGFR and K-ras G12C mutation (18, 31). The Ad-TTS/Bax vector also induced significant cell death in these cells (Fig. 4D). Therefore, these results suggest that Ad-TTS/Bax induces exogenous BAX expression and cell death in pulmonary adenocarcinoma cells including gefitinib-resistant cells.


Figure 4
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  		Figure 4. Pulmonary adenocarcinoma–specific apoptosis induced by Ad-TTS/Bax. A, schematic representation of Ad-TTS/GFP or Ad-TTS/BAX. B, detection of TTF1 and Bax expression induced by Ad-TTS/Bax. Cells were treated with the same dosage of Ad-TTS/GFP or Ad-TTS/Bax as described in Materials and Methods. Thirty-six hours after infection, expressions of TTF1 and BAX were analyzed by immunoblot. Exogenous HA-BAX protein expression was detected with antihemagglutinin antibody. C, flow cytometric analysis of apoptosis induced by induced by Ad-TTS/Bax. Cells were infected with Ad-TTS/GFP or Ad-TTS/Bax for 72 h and sub-G0-G1 DNA content was measured by propidium iodide staining and flow cytometric analysis. D, detection of apoptosis induced by Ad-TTS/Bax in gefitinib-resistant pulmonary adenocarcinoma A549, H441, and H358 cells. Cells were infected with Ad-TTS/GFP or Ad-TTS/Bax for 72 h and subdiploid DNA was measured by propidium iodide staining and flow cytometric analysis.

 
TTS/Bax Induced Minimal Cell Death in Stem Cells
The drawback of using the cytomegalovirus (CMV) promoter or the hTERT (a telomerase component) promoter for cancer gene therapy is that their activities are high not only in cancer cells but also in normal stem cells and progenitor cells. To determine the safety of the TTS promoter relative to the CMV promoter or the hTERT promoter, the sub-G0-G1 populations were measured by flow cytometry after transfection with the plasmids expressing Bax driven by the CMV promoter (pCMV/Bax), the hTERT promoter (phTERT/Bax), or the TTS (pTTS/Bax; Fig. 5 ). The CMV promoter (pCMV/Bax) or the hTERT promoter (phTERT/Bax) was active enough to induce BAX expression (Fig. 5A) and cell death in both embryonic stem cells (27.22–27.63%) and mesenchymal stem cells (18.22–27.37%; Fig. 5B). However, the TTS promoter (pTTS/Bax) caused little BAX expression and cell death in both stem cells (1.16–4.73%). These results show that the TTS system induces the proapoptotic gene only in the cancer cells but not in normal cells including stem cells, whereas cancer gene therapy systems using the CMV promoter or the hTERT promoter induce the expression of their therapeutic gene not only in cancer cells but also in stem cells.


Figure 5
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  		Figure 5. Comparison of BAX expression and apoptosis induced by CMV, hTERT, or TTS promoter in embryonic stem cells and mesenchymal stem cells. A, nucleofection was done in embryonic stem cells and human mesenchymal stem cells with pCMV/Bax (4 µg), phTERT/Bax (4 µg), or pGL3.hSPA1-157 5x/BAX (pTTS/Bax; 4 µg). Thirty-six hours after transfection, expressions of TTF1 and BAX were analyzed by immunoblot. Exogenous HA-BAX protein expression was detected with antihemagglutinin antibody. B, detection of apoptosis induced by the plasmid expressing Bax driven by CMV, hTERT, or TTS promoter after nucleofection. Four micrograms of indicated plasmid vector were nucleofected into the cells. Seventy-two hours after transfection, subdiploid DNA was measured by propidium iodide staining and flow cytometric analysis.

 
Glucocorticoid Inhibition of Cell Death by the TTS/Bax System
An important feature of the TTS system is that it contains the two negative glucocorticoid-responsive elements of the hSPA1 promoter (Figs. 1 and 4A) to control the treatment. We therefore examined the effect of dexamethasone (a glucocorticoid) on the TTS/Bax system. Western blot and flow cytometric analysis showed that dexamethasone reduced Bax expression (Fig. 6A ) and caspase-3 activity (Fig. 6B) in pulmonary adenocarcinoma A549 cells. Hoechst staining showed that dexamethasone also inhibited apoptosis induced by Ad-TTS/Bax (Fig. 6C).


Figure 6
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  		Figure 6. Dexamethasone decreased BAX expression and apoptosis induced by Ad-TTS/Bax. A, A549 cells were treated with dexamethasone (Dex; 0–500 nmol/L) for 36 h after Ad-TTS/Bax infection. HA-BAX expression was analyzed by immunoblot with antihemagglutinin antibody. B, dexamethasone inhibition of caspase-3 activation mediated by Ad-TTS/Bax. For caspase inhibition, A549 cells were treated with dexamethasone (500 nmol/L) or z-VAD-fmk (50 µmol/L) for 24 h after Ad-TTS/Bax infection. C, 72 h after Ad-TTS/Bax infection, A549 cells treated with (+) or without (–) dexamethasone (500 nmol/L) were stained with Hoechst-33342 dye.

 
Discussion

The advantage of cancer gene therapy is that a proapoptotic gene is expressed only in the targeted cancer cells by using a cancer-specific promoter. The proapoptotic genes, including Bax, are able to induce apoptosis in various kinds of cancer cells and p53-sensitive/resistant or gefitinib-sensitive/resistant cancer cells (32–37). The activity of the cancer-specific promoter (e.g., hTERT promoter) is often high in normal stem cells, but the combination of the cancer-specific promoter with the tissue-specific promoter enables the promoter activity to be specific only to cancer cells and not to normal stem cells (13). Hence, we further developed a system (TTS/Bax) that induces BAX expression and cell death only in pulmonary adenocarcinoma cells and not in normal cells including stem cells. Moreover, the negative glucocorticoid-responsive element in the system allows it to be drug controllable by dexamethasone. The approach that we used to make the TTS system can be applied to treat other types of cancer. In our system, we used the transcription factor TTF1 and the promoter of surfactant protein A1 (hSPA1) to make the system pulmonary adenocarcinoma specific. TTF1 was detected in 76% of pulmonary adenocarcinomas (38). hSPA1 is a lung-specific marker and synthesized by alveolar type II cells or Clara cells (39). Gazdar et al. (40) analyzed 41 kinds of lung cancer cells and reported that cytoplasmic structure characteristic of type II cells or Clara cells was detected in 41% of pulmonary adenocarcinoma while rare in small and large-cell carcinoma and squamous cell carcinoma of the lung. Our screening of TTF1 and hSPA1 gene expressions in various cancer cells (Fig. 2) agrees with previous results (38, 40). Thus, expressions of TTF1 and hSPA1, as well as those of markers for alveolar type II cells or Clara cells, are relatively high in pulmonary adenocarcinoma. The use of the hSPA1 promoter under the hTERT promoter–driven TTF1 is adequate to target pulmonary adenocarcinoma. Recent microarray analysis in different types of cancer cells has provided each cancer type specific genes (41, 42). For example, introducing a mesothelioma-specific transcription factor and its target promoter into the tissue- and cancer-specific promoter system creates a mesothelioma-specific promoter system that can selectively kill mesothelioma.

Gene transduction technology using adenovirus vector for gene therapy has recently been improved. For example, adaptor-based adenovirus targeting technique has been well developed (43). Using the TTS/Bax promoter system with the transduction-improved adenovirus vector will contribute to the efficacy and safety of pulmonary adenocarcinoma gene therapy. If a virus vector cannot be used because of uncertainty about unpredictable side effects (44), a liposome-DNA delivery system can be used. Liposome-DNA delivery is considered to be a safer and less toxic delivery system than the virus delivery system (45, 46). Instead of the adenovirus vector, the use of the liposome delivery system with the TTS/Bax plasmid can be applied to treat pulmonary adenocarcinoma.

One interesting aspect of the molecular mechanism of the TTS system is the involvement of additional transcriptional factors and/or cofactors that are specific to pulmonary adenocarcinoma cells. TTF1 expression was enough to induce the TTS promoter activity in pulmonary adenocarcinoma cells including TTF1-negative cells. However, TTF1 expression was not enough to induce the TTS promoter activity in other cancer cells including TTF1-positive cells. TTF1 interacts with transcription factors and coactivators on the SPA promoter (27). TTF1 is also shown to form a protein complex (47). The known and unknown interacting partners of TTF1 that are expressed only in pulmonary adenocarcinoma might determine the gene regulation of the TTS system. Identification of the TTF1-interacting protein complex in pulmonary adenocarcinoma will be useful to understand TTF1 activation and construct a better pulmonary adenocarcinoma–specific promoter system.

Cancer gene therapy using a tissue- and cancer-specific promoter system, such as TTS/Bax, which forcefully induces proapoptotic gene expression and cell death in drug-resistant cancer cells but not in normal cells, is a much-needed advancement in cancer gene therapy. The combination of such a carefully designed promoter system with safer and better transduction systems may provide a new model of cancer gene therapy.

Acknowledgments

We thank H. Yamanishi for technical advice and J. Whitsett for critical reading of the manuscript.

Footnotes

Grant support: Ministry of Education, Science, and Culture and Ministry of Health and Welfare, Japan.

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

Received 7/14/06; revised 9/19/06; accepted 11/10/06.

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