Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice

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Abstract

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

Introduction

Long-term survival of lung cancer patients treated with conventional therapies remains poor; thus, the need for novel approaches is raised. Gene delivery through inhalation may provide a means of treatment for a wide range of pulmonary disorders and offer numerous advantages over other invasive modes of delivery. Several studies have demonstrated that binding of DNA with cationic polypeptides such as polylysine, polythylenimine (PEI), protamine and histone may be useful for gene delivery both in vivo and in vitro.1 Among these polypeptides, PEI has received the most attention as a carrier of gene delivery due to its stability during nebulization. However, practical application of PEI has been limited because of its potential toxicity caused by the characteristic accumulation of polycations. Therefore, we have tried to improve the performance of PEI as a gene carrier in terms of binding to cell surface, endocytosis, escape from endosomal lysosomal network, translocation to the cell nucleus, and vector unpacking. Toward this end, we have developed glucosylated PEI (GPEI) that showed low toxicity with high efficiency due to glucose moiety as a carrier.2

Programmed cell death (PDCD4), also termed apoptosis, plays a fundamental role in many biological processes such as embryogenesis, normal turnover and immune homeostasis.3 Although a number of caspase-dependent and -independent apoptotic pathways have been identified, the mechanisms of apoptosis are very complex and are still largely unknown.4 PDCD4 is highly conserved during evolution indicating an important biological function of this protein. PDCD4 has two conserved α-helical MA-3 domains, which are known to be involved in protein–protein interactions.5 PDCD4 is also known as a novel eIF4A-binding partner and to inhibit the helicase activity of eIF4A, subsequently inhibiting cap-dependent translation.6 PDCD4 also binds eIF4G, independently of its interaction with eIF4A although the consequence of PDCD4 binding to eIF4G is not well understood. PDCD4 seems to be the first example of a protein in mammalian cells that inhibits translation through attenuation of eIF4A activity.7

The transcription factor activator protein-1 (AP-1) can be produced by 18 different dimeric combinations of protein from the Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) families.8 AP-1 regulates a variety of cellular processes, including proliferation, differentiation and apoptosis.9 Recently, PDCD4 has been demonstrated to inhibit the synthesis or activation of AP-1 protein.10 In this study, aerosol containing PDCD4 was delivered into AP-1 reporter mice to demonstrate that in vivo aerosol PDCD4 delivery can be an effective approach for suppressing AP-1 activity. Here, we report that aerosol-delivered PDCD4 could control processes related to apoptosis in the lungs of AP-1 luciferase reporter mice. Our results confirmed the hypothesis that suppression of AP-1 activity by PDCD4 may provide a useful tool for designing lung tumor prevention and treatment strategy by which PDCD4 functions as a transformation suppressor.

Results

Delivery efficiency of GPEI/PDCD4 complex

Aerosol delivery efficiency of GPEI/green fluorescent protein (GFP) was confirmed by immunofluorescence assay. Analysis of mouse-specific intracellular antigen against macrophage and monocyte indicated that most of the GFP expression plasmid was delivered successfully into lung tissues. Although some GFP plasmid was ingested by phagocytosis (shown as red color), most was delivered into pulmonary cells (Figure 1). Western blot analysis confirmed successful aerosol delivery of wild-type (WT) PDCD4 to the lungs. PDCD4 WT delivery resulted in a statistically significant increase in PDCD4 protein levels relative to control and vector control (supporting information, P<0.001, n=6). Delivery of mutant PDCD4 also resulted in significantly higher PDCD4 expression relative to control and vector control, although the expression of mutant PDCD4 appears to be slightly less than that of PDCD4 WT (Figures 2a and b; supporting information, P<0.05, n=6). Immunohistochemistry analysis distinctively confirmed that aerosol PDCD4 delivery in the lung worked properly in agreement with the western blot result. As shown in Figure 2c, PDCD4 WT increased the expression of PDCD4 proteins in the lung tissues (right panel), and such increment of PDCD4 expressed was also found in PDCD4 mutant group with slightly less expression compared to WT (middle panel). However, such increased PDCD4 expression was not observed in vector control (left panel). Together with previous works showing high transfection efficiency by aerosol gene delivery,2, 11, 12 we could confirm that aerosol delivery of GPEI/PDCD4 complexes was able to increase PDCD4 protein levels in the lungs of AP-1 luciferase reporter mice.

Figure 1
figure1

Efficiency of aerosol PDCD4 delivery. Delivery efficiency of GPEI as a gene carrier was evaluated using GPEI/GFP complex. AP-1 luciferase reporter mice were exposed to aerosol containing GPEI/GFP complex for 30 min, and the mice were killed after 48 h. Immunohistochemical analysis of mouse-specific intracellular antigen against macrophage and monocyte in GPEI/pcDNA3.1-GFP (a and b) and GPEI/pcDNA3.1-delivered (c and d; control) lungs. Red signals (a and c) indicate ingestion of some GFPs by alveolar macrophages and monocytes. Green signals (b) indicate that most of the delivered GFPs were transfected into lung tissues. Although some GFPs were ingested by phagocytosis, most were transfected into pulmonary cells. Magnification, × 400, scale bar, 20 μm. Pictures were taken by confocal laser scanning microscope.

Figure 2
figure2

Effect of aerosol-delivered PDCD4. Expression levels of PDCD4 proteins in the PDCD4 delivered lungs. (a) Western blot analysis. Lysates from the lungs AP-1 luciferase reporter mice treated with aerosol containing PDCD4 WT or mutant PDCD4 were analyzed for PDCD4 protein expression by western blot. (b) Densitometric analysis. The bands-of-interests of western blot were analyzed further by densitometer. Data were normalized to actin (mean±s.e., n=3). **P<0.01, significantly different from vector control. *P<0.05, significantly different from vector control. (c) Immunohistochemical analysis of PDCD4 protein expression. Lungs were fixed and incubated with PDCD4 antibody. Dark brown represents positive signal. PDCD4 is highly expressed in PDCD4 delivered mouse lung compared to vector control. Magnification, × 200; scale bar, 100 μm. WT, PDCD4 WT; D418A, PDCD4 mutant type. WT, wild type.

Aerosol delivery of PDCD4 facilitated apoptosis

The effects of aerosol PDCD4 delivery on apoptotic signals were investigated. Aerosol-delivered PDCD4 WT significantly increased proapoptotic proteins such as Bcl-associated death promoter (BAD), Bcl2-associated X protein (BAX) and BH3 interacting domain death agonist (BID). Aerosol delivery of PDCD4 also significantly decreased antiapoptotic BCL-XL protein level. However, such findings were not demonstrated by the aerosol delivery of PDCD4 mutant (D418A) (Figure 3a; P<0.05, n=3). Densitometric analysis of western blot could re-confirm that aerosol delivery of PDCD4 was able to facilitate apoptosis in the lungs of AP-1 luciferase reporter mice (supporting information). Note that such effects of PDCD4 WT were not observed in PDCD4 mutant D418A (Figure 3b). Moreover, as shown in TdT-mediated dUTP nick end labeling (TUNEL) assay (arrows in Figure 3c) and quantitative analysis of TUNEL-positive cells (Figure 3d; P<0.001, n=3), PDCD4 WT significantly increased the apoptosis compared to vector control (left panel of Figure 3c; P<0.001, n=3) or PDCD4 mutant form (middle panel of Figure 3c).

Figure 3
figure3

Effect of aerosol-delivered PDCD4 on proapoptotic and antiapoptotic proteins. (a) Western blot analysis. Lysates from the lungs of AP-1 reporter mice treated with aerosols containing PDCD4 WT or mutant were analyzed for protein levels of BID, BAD, BAX and BCL-XL by western blot. (b) Densitometric analysis. The bands-of-interest of western blot were further analyzed by densitometer. *P<0.05, significantly different from vector control. Data were normalized to actin (mean±s.e., n=3). (c) TUNEL assay. Apoptotic signals (arrows) were clearly detected in the lungs of AP-1 mice treated with aerosol-delivered PDCD4 WT compared to vector control and PDCD4 mutant. Scale bar, 100 μm (d) Quantitative analysis of TUNEL-positive cells. TUNEL-positive cells were counted and then graphed. Data are shown as mean±s.e. **P<0.01, significantly different from vector control. VEC, vector control; D418A, PDCD4 mutant; PDCD4, WT PDCD4.

Aerosol delivery of PDCD4 suppressed signals important for cell-cycle control

We determined whether aerosol delivery of PDCD4 had any effect on the cell-cycle proteins. Aerosol-delivered PDCD4 WT significantly decreased the protein expression levels of PCNA, cyclin D1 and CDK4 protein in the lungs of AP-1 luciferase reporter mice compared with those of vector control and PDCD4 mutant (Figure 4a). Again such findings were supported further by densitometric analysis of western blot (Figure 4b, supporting information; P<0.05 n=3). Immunohistochemical study of PCNA clearly supported that increased levels of PDCD4 WT suppressed PCNA protein expression in the lung (Figure 4c).

Figure 4
figure4

Expression levels of cyclin D1, CDK4 and PCNA in the PDCD4 aerosol-delivered lungs. (a) Western blot analysis. Lysates from the lungs of AP-1 mice treated with aerosol-delivered PDCD4 WT or mutant were analyzed for protein levels of, cyclinD1, CDK4, PCNA by western blot. (b) Densitometric analysis. The bands-of-interest of western blot were further analyzed by densitometer. *P<0.05, significantly different from vector control. #P<0.05, significantly different from corresponding PDCD4 WT. Data were normalized to actin (mean±s.e., n=3). (c) Immunohistochemical analysis of PCNA. Lungs from vector control, PDCD4 mutant and PDCD4 WT-delivered mice were fixed and incubated with PCNA antibody. Dark brown represents positive signal. PCNA was less expressed in PDCD4 WT delivered mouse lung compared to vector control and PDCD4 mutant. Magnification, × 200; scale bar, 100 μm. PDCD4, PDCD4 WT; D418A, PDCD4 mutant type.

Aerosol delivery of PDCD4 inhibited AP-1 activity

In agreement with previous results demonstrating PDCD4 specifically inhibits AP-1 activity in vitro,10 we analyzed the effects of PDCD4 in vivo on AP-1 luciferase activity of the reporter mice pretreated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Our result showed that aerosol delivery of PDCD4 WT inhibited TPA-induced AP-1 luciferase activity, relative to control, vector control and PDCD4 mutant (Figure 5; P<0.001, n=3). To investigate potential changes in protein expression of members of the AP-1 family of transcription factors, western blot analysis was carried out. Expression levels of c-Jun, Fra-1, Jun D were decreased relative to controls by the aerosol delivery of PDCD4 WT while c-Fos protein expression remained unchanged (Figure 6a). Although the change of expression of individual members of the AP-1 family, except c-Jun, was not statistically significant by densitometric analysis (Figure 6b, supporting information), the overall results of PDCD4 WT expression was a general suppression of TPA-induced overexpressed AP-1 activity (Figure 5).

Figure 5
figure5

Inhibition of AP-1 luciferase activity by PDCD4. AP-1 luciferase reporter mice were given by i.p. injection with TPA at 5 nmol in 200 μl PBS, four times per 2 days, then, GPEI/PDCD4 complexes were inhaled into the mice. Pulmonary luciferase activity was measured after 48 h. **P<0.01, significantly different from TPA control mice. ##P<0.01, significantly different from corresponding PDCD4 WT. Data are representative of three independent experiments (mean±s.e., n=3).

Figure 6
figure6

Expression levels of AP-1 family proteins in the PDCD4 aerosol-delivered lungs. (a) Western blot analysis. Fra-1, c-Jun and Jun-D expression levels were decreased by PDCD4 delivery. Lysates from the lungs of AP-1 mice treated with aerosol-delivered PDCD4 WT or mutant were analyzed for protein levels of Fra-1, c-Jun and Jun-D by western blot. (b) Densitometric analysis. The bands-of-interest of western blot were further analyzed by densitometer. *P<0.05, significantly different from vector control. Data were normalized to actin (mean±s.e., n=3). PDCD4, PDCD4 WT; D418A, PDCD4 mutant type.

Discussion

The long-term survival of lung cancer patients treated with surgery, radiation therapy and chemotherapy remains poor and has changed little in decades. The need for novel approaches remains high and the delivery of genes by inhalation holds promise for the treatment of a wide range of pulmonary disorders. There was great interest in the delivery of genes directly to the lungs by aerosol, and most early efforts focused on the use of non-viral vectors, particularly cationic lipids.13 In this study, we used GPEI which showed low toxicity due to glucose moiety proven by previous studies.2, 11, 12 In fact, the current study also confirmed that GPEI-mediated delivery of PDCD4 was successful because of satisfactory expression of GFP protein in the lungs of AP-1 reporter mice as shown in Figure 1.

Growing evidences has demonstrated that apoptosis and cell-cycle pathways are contributing factors in lung cancer.14 Efficient inhibition of growth and/or promotion of apoptosis of cancer cells are promising targets for prevention and treatment of lung cancer. Apoptosis is regulated by a variety of proapoptotic and antiapoptotic factors. Activations of BAX and BAD are known to induce the apoptosis in lung cancer cells.15 Several studies have shown that overexpression of BCL-XL prevents the mitochondrial release of cytochrome c, thereby facilitating the apoptosis of human non-small cell lung cancer cells.16 In this study, we investigated whether aerosol delivery of PDCD4 could affect apoptosis signaling pathway. Our findings showed that aerosol delivery of PDCD4 WT decreased antiapoptotic protein BCL-XL, but increased the proapoptotic proteins such as BAX, BAD and BID (Figures 3a–c). Together, aerosol delivery PDCD4 could facilitate the apoptosis in the lungs of AP-1 luciferase reporter mice.

Deregulation of genes involved in the cell cycle is a hallmark of a variety of human cancers, including non-small cell lung cancer.17 Therefore, the use of compounds capable of targeting aberrantly expressed cell cycle-related proteins may be a promising approach to treatment of lung cancer. Cyclin D1 works transiently during the G1 phase and can be activated by CDK4. The complex of cyclin D1 and CDK4 further phosphorylates the key cell-cycle regulator protein pRB. Together, unregulated phosphorylation of pRB by activated CDK4 in response to overexpressed cyclin D1 may lead to loss of growth control.18 Our study demonstrated that aerosol delivery of PDCD4 decreased cyclin D1 and CDK4 protein expression significantly (Figures 4a–c), suggesting that the aerosol delivery of PDCD4 can cause cell-cycle arrest through downregulation of the cyclin D1/CDK4 complex formation.

The AP-1 family of transcription factors acts as environmental biosensors to various external toxic stimuli and regulates the expression of genes involved biological processes. For example, several lines of evidences have shown that asbestos may cause lung cancer through abnormal AP-1 activation in rat lung tumors and that Fra-1 may play an active role in this tumorigenesis.19 Our western blot results showed that expression levels of c-Jun, Fra-1, Jun D were generally decreased by the aerosol delivery of PDCD4 WT (Figure 6a). Although a statistically significant change was only observed in c-Jun based on densitometric analysis (Figure 6b), the overall results of PDCD4 WT expression was a general suppression of TPA-induced AP-1 activity (Figure 5). Together, aerosol delivery of PDCD4 may provide very promising target for the prevention and treatment of lung cancer. In fact, recent studies have shown that the c-Jun oncogene is frequently overexpressed in non-small cell lung cancer, and dysregulation of c-Jun may be involved in the process of human lung carcinogenesis.20 Moreover, another line of evidence has also shown that AP-1 protein, Fra-1 is required for the activation of AP-1-dependent transcription, suggesting that Fra-1 activation may be necessary for tumor promotion.21 Results reported here suggest that PDCD4 delivery results in a consistent decrease of Fra-1 protein levels relative to control, vector control and D418A. Therefore, targeting c-Jun as well as Fra-1 by PDCD4 delivery into the lung may be beneficial for lung cancer prevention during either tumor promotion or tumor progression. Together, our results have demonstrated that there have been strong correlation between the level of PDCD4 protein expression, the degree of apoptosis and suppression of AP-1 in the lungs of mice exposed to delivered PDCD4.

In conclusion, our results clearly demonstrated that aerosol delivery of GPEI/PDCD4 complex could facilitate apoptosis, regulate cell cycle and suppress AP-1 activity. Together with the unique and unusual characteristics of PDCD4, which works by targeting translation, our study strongly suggest that aerosol gene delivery may provide an effective non-invasive model of gene delivery and aerosol delivery PDCD4 may provide a target for lung cancer prevention as well as treatment in the future.

Materials and methods

Materials

BID, BAD, BAX, BCL-XL, c-JUN, c-FOS, JunD, JunB, cyclin D1, CDK4, PCNA and actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase antibodies were obtained from BD Biotechnology (San Jose, CA, USA). TPA was purchased from Sigma-Aldrich (St Louis, MO, USA). Monoclonal PDCD4 antibody was produced using a general method described elsewhere. pcDNA3.1-GFP was purchased from Invitrogen (Carlsbad, CA, USA).

Preparation of GPEI/PDCD4 complex

GPEI/DNA complexes were prepared at 1 μg of DNA to 2.67 μg of GPEI carrier according to the previous report.2, 11, 12 Briefly, self-assembled GPEI/PDCD4 complexes were initiated in distilled water by adding 1 mg of pcDNA3.0-PDCD4 or GFP or pcDNA3.0-PDCD4 mutant plasmid to GPEI, drop by drop, under gentle vortexing, and the final volume was adjusted to 50 ml. The complexes were then incubated at room temperature for 30 min before use. Our gene delivery systems have been validated by several studies in our group.2, 11, 12

In vivo aerosol delivery of GPEI/PDCD4 complex

Experiments were carried out on 6-week-old AP-1 luciferase reporter mice, a mixed strain of C57BL/6 and DBA/N2.9 Breeding AP-1 reporter mice were obtained from Human Cancer Consortium-National Cancer Institute (Frederick, MD, USA) and kept in the laboratory animal facility with temperature and relative humidity maintained at 23±2°C and 50±20%, respectively, and were kept on a 12-h light/dark cycle. Mice were placed in nose-only exposure chamber (NOEC) and exposed to the aerosol. Aerosol was generated using the patented nebulizer (Korea patient no. 20304964) designed to minimize sample loss as well as shearing force. Complex solution contained 1 mg of pcDNA3.1-PDCD4 WT or pcDNA3.1-PDCD4 mutant plasmid DNA or GFP. Mice were placed in NOEC, and GPEI/PDCD4 or GFP complexes were aerosolized using the nebulizer for 30 min. WT of PDCD4 (PDCD4 WT) and mutant type PDCD4 (D418A) plasmids were described in previous works. In the works, PDCD4 mutant D418A (Asp to Ala at amino acid 418) clearly demonstrated to fail to bind eIF4A and therefore fail to regulate translation.22 Two days after exposure, the mice were killed, and lung samples were collected for further analysis.

Western blot analysis

The lungs were homogenized with a lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM ethylenediaminetetraacetic acid; Promega, Madison, WI, USA) and protein was measured using the Bradford kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on SDS-PAGE and transferred into nitrocellulose membranes (Amersham Pharmacia, Cambridge, UK). After membranes were blocked in TTBS containing 5% skim milk for 1 h, immunoblotting was performed by incubating overnight 4°C with corresponding primary antibodies in 5% skim milk and then with second antibodies conjugated to horseradish peroxydase (HRP) for 2 h at room temperature or overnight at 4°C. After washing, the bands-of-interests were pictured by luminescent image analyzer LAS-3000 (Fujifilm, Japan). Results were quantified using a measure program of LAS-3000.

AP-1 luciferase assay

AP-1 luciferase reporter mice were given by intraperitoneally (i.p.) injection with TPA at 5 nmol in 200 μl PBS four times per two days. Mice were placed in NOEC, and GPEI/PDCD4 complexes were aerosolized using the nebulizer for 30 min. Two days after exposure, the mice were killed, and lung samples were collected. The luciferase activities were measured in tissue extracts. The lungs were homogenized in passive lysis buffer (Promega, Madison, WI, USA). The homogenates were centrifuged for 20 min at 4500 r.p.m. at 4°C, the supernatant was further centrifuged for 15 min at 1300 r.p.m. at 4°C. Luciferase activities were measured using the luciferase assay kit (Promega).

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue section were cut at 5 μm and transferred to plus slides (Fisher Scientific, Pittsburgh, PA, USA). The tissue were deparaffinized in xylene and rehydrated through alcohol gradient. The tissue sections were incubated in 150 μl proteinase K, washed and incubated in 0.3% hydrogen peroxide (AppliChem, Darmstadt, Germany) for 30 min to quench endogenous peroxidase activity. After washing in PBS, the tissue sections were 3% BSA in PBS for 1 h at room temperature to block the unspecific binding sites. Primary antibodies were applied on tissue section for overnight 4°C. The following day, tissue sections were washed and incubated with secondary HRP-conjugated antibodies for 2 h at room temperature. After washing, tissue sections were counterstained with Mayer's hematoxylin (Dako, Caepinteria, CA, USA), and the slides were reviewed using a light microscope (Carl Zeiss, Thornwood, NY, USA).

TUNEL assay

Formalin-fixed, paraffin-embedded lung tissue slides were deparaffinized in xylene and rehydrated through alcohol gradient. The slides were washed with PBS, and nicked DNA ends were labeled by terminal TUNEL method using in situ cell death detection kit (Roche, Basel, Switzerland) following the manufacturer's protocol. As a final step, tissue sections were counterstained with methyl green (Trevigen, Gaithersburg, MD, USA). TUNEL-positive cells were counted under microscope for quantitative analysis.

Data analysis

Quantification of Western blot analysis was performed using Multi Gauge version 2.02 program (Fujifilm). All results are expressed as means±s.e. The results were analyzed by Student's t-test (Graphpad Software, San Diego, CA, USA). *P<0.05 was considered significant and **P<0.01 highly significant compared with corresponding vector control values. #P<0.05 was considered significant and ##P<0.01 highly significant compared with corresponding values from PDCD4 WT.

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Acknowledgements

This work was partially supported by the grants from the KOSEF (R01-2005-000-10087-0) of the Ministry of Science and Technology in Korea. MHC was supported by the Nano Systems Institute-National Core Research Center (NSI-NCRC) program of KOSEF. SKH, JH, THK are also grateful for the award of the BK21 fellowship. KHL was supported by 21C Frontier Functional Human Genome Project (FG03-0601-003-1-0-0) and National Nuclear R&D Program from Ministry of Science and Technology. GRB Jr was supported by National Cancer Institute Grant CA84573.

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Correspondence to M-H Cho.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Hwang, S., Jin, H., Kwon, J. et al. Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice. Gene Ther 14, 1353–1361 (2007) doi:10.1038/sj.gt.3302983

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Keywords

  • programmed cell death 4
  • apoptosis
  • cell cycle
  • activator protein-1
  • non-invasive aerosol gene delivery

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