Mol Cell Biochem (2013) 375:1–9 DOI 10.1007/s11010-012-1484-7
Effect of tumor suppressor PTEN gene on apoptosis and cell cycle of human airway smooth muscle cells Liang Luo • Yuan Qi Gong • XieFei Qi • WenYan Lai • Haibing Lan • Yaling Luo
Received: 19 June 2012 / Accepted: 17 October 2012 / Published online: 29 December 2012 Ó Springer Science+Business Media New York 2012
Abstract It is well established that hyperplasia and decreased apoptosis of airway smooth muscle cells (ASMCs) play an important role in the asthmatic airway remodeling. Tumor suppressor PTEN gene with phosphatase activity plays an important regulatory role in embryonic development, cell proliferation, and apoptosis, cell cycle regulation, migration (invasion) of the cytoskeleton. We hypotheses that PTEN gene could affect the growth and viability of ASMCs through the regulation of PI3K/ Akt, MAPK, and cell cycle-related gene expression. We constructed a recombinant adenovirus to transfect ASMCs. Cells were divided into the overexpression of PTEN gene group (Ad-PTEN-GFP), negative control group (Ad-GFP), and blank control group (DMEM). The cell apoptosis of
ASMCs were evaluated by Hoechst-33342 staining and PE-7AAD double-labeled flow cytometry. The cell cycle distribution was observed by flow cytometry with PI staining. The expression of PTEN, p-Akt, total-Akt, p-ERK1/2, total-ERK1/2, cleaved-Caspases-3, Caspases-9, p21, and Cyclin D1 were tested by the Western blotting. Our study revealed that overexpression of PTEN gene did not induce apoptosis of human ASMCs cultured in vitro. However, overexpression of PTEN inhibited proliferation of human ASMCs cultured in vitro and was associated with downregulation of Akt phosphorylation levels, while did not affect ERK1/2 phosphorylation levels. Moreover, overexpression of PTEN could induce ASMCs arrested in the G0/G1 phase through the downregulation of Cyclin D1 and upregulation of p21 expressions.
The authors Liang Luo and Yuan Qi Gong contributed equally to this study.
Keywords PTEN gene Airway smooth muscle cells Cell apoptosis Cell cycle
L. Luo Department of Medical Intensive Care Unit, The First Affiliated Hospital of Sun Yat-sen University, No. 58, Zhong Shan Er Road, Guangzhou, People’s Republic of China Y. Q. Gong X. Qi H. Lan (&) Department of Intensive Care Unit, The Second Affiliated Hospital of NanChang University, Nanchang, People’s Republic of China e-mail:
[email protected] W. Lai Laboratory of Cardiovascular Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China Y. Luo (&) Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, No. 1838, Guangzhou North Avenue, Guangzhou, People’s Republic of China e-mail:
[email protected]
Introduction The morbidity and mortality of bronchial asthma, one of most common chronic diseases, continue to increase in the past 20 years. Economic burden incurred by asthma have surpassed the total cost of lung tuberculosis and AIDS [1]. A recent study reported that the total cost attributed to asthma in the United States was $56 billion [2]. Although the prevalence of bronchial asthma is lower in China than developed countries, the mortality rate is up to 36.7/ 100,000, ranking the first in the world [3]. Therefore, bronchial asthma has become a serious worldwide public health burden and social problem. It is urgent to find more effective treatment approaches to alleviate the symptoms.
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However, anti-inflammatory treatments could not eradicate asthma due to incomplete elimination of airway hyper responsiveness. Thus, the association between airway inflammation and hyper responsiveness is unknown [4]. Asthma is characterized by airway remodeling that linked between airway inflammation and airway hyper responsiveness. Asthmatic airway remodeling is airway wall structural changes including epithelial damage, basement membrane thickening, subepithelial fibrosis, goblet cells, and submucosal gland hypertrophy, smooth muscle cell hyperplasia, hypertrophy, and angiogenesis [5]. The airway smooth muscle cells (ASMCs), one of the main structural components of airway remodeling, are not only the target cell of airway inflammation but also the effect cell participating in asthmatic pathological process (airway inflammation, airway remodeling, and hyper responsiveness) and disease aggravation. The hyperplasia, hypertrophy, and migration of ASMCs in the airway in asthma not only make more intense bronchial contractile response to stimulation, increased airway hyper responsiveness, and ASMCs proliferation promotes airway wall thickness but also result in increased basic airway resistance and irreversible airway obstruction [6]. As a kind of immune regulator cells in asthma, ASMCs continuously produce cytokines including IL-6, TGF-b, ICAM-1, matrix metalloproteinases (MMPs), chemokine factors, adhesive factors, and growth factors, among which it have been substantiated that MMP-1, MMP-9, and MMP-12 play an important role in airway remodeling [7]. Therefore, ASMCs are recently regarded as target of research and treatment in asthma by more and more scholars [8–10]. The phosphatase and tensin homology deleted on chromosome ten (PTEN) located at chromosome 10q 23.3, 200 kb, was first reported by three study groups in 1997. The phosphatase and tensin homology deleted on chromosome ten gene is the first tumor suppressor with both anti-cancer and phosphatase activity and plays an important regulatory role in embryonic development, cell proliferation, and apoptosis, cell cycle regulation, migration (invasion), and the cytoskeleton [11]. The phosphatase and tensin homology deleted on chromosome ten protein can regulate PI3K/PKB/Akt signaling cascades in the PI-3kinase pathway, Ras-Raf-MEK1/2-ERK1/2 signaling cascades in the MAPK pathway, and also can specifically induce expression of cyclin-dependent kinase inhibitor p21 and p27 etc. [12]. These messenger factors are the primary signaling factors mediating ASMCs growth, viability, and anti-apoptosis. As indicated in previous studies, PTEN gene has an effect on tumor cells via regulating expression of PI3K, MAPK, and cell cycle-related protein. It affects the proliferation and viability of tumor cells, vascular smooth muscle cells, and fibroblasts. We supposed that PTEN had
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similar effect on ASMCs. We used gene overexpression carried by adenovirus vectors, flow cytometry, Hoechst33342 staining, CCK-8 method, and immunoblotting in our study. Effect of PTEN gene overexpression on cell apoptosis and cell cycles in human ASMCs cultured in vitro was assessed. The regulation mechanism of Akt, ERK1/2, Cyclin D1, cyclin-dependent kinase inhibitor p21 by PTEN gene was preliminary investigated. It was expected to provide further knowledge of asthmatic airway remodeling and contribute to better control of asthma.
Materials and methods Cell culture Human ASMCs were isolated from the lobar or main bronchus obtained from lung resection donors, using procedures approved by the Division of Thoracic Surgery and the Human Ethics Committee of Nanfang Hospital, Southern Medical University. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin (all from Gibco, USA). Cells from passages 3–8 were used for the experiments. Immunocytochemical characterization The cells were subcultured and grown to near confluence. After washing twice with ice-cold phosphate buffered saline (PBS), the cells were suspended in ice-cold 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100. They were washed and subsequently incubated with anti-a smooth muscle actin antibody (Bios, Beijing, China). After incubation, the cells were washed twice with PBS and further incubated with goat anti-rabbit IgG (Bios). The positive specimens were visualized using a microscope and then photographed. Adenovirus construction and infection Recombinant adenoviruses encoding human wild-type PTEN cDNA (Ad-PTEN) and a control virus containing no cDNA insert [empty virus, GFP-labeled adenovirus vector (Ad-GFP)] were constructed using the pAdxsi system, and the viruses were amplified as described previously (21). Plasmids carrying wild-type PTEN cDNA gene (p-WTPTEN) and empty plasmids (p-eGFP) were generous gifts from Professor Kenneth M Yamada at the National Institutes of Health. Airway smooth muscle cells were grown in 25-cm2 culture flasks in DMEM containing 10 % FBS. When the cells were grown to 70 % confluence, the
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medium was changed to DMEM containing no FBS, and viruses were added to the medium at a multiplicity of infection (MOI) of 100 and incubated at 37 °C, 5 % CO2 for 4 h. Then the culture medium with 10 % FBS was added. After 18 h, the medium was changed to fresh DMEM containing 10 % FBS, followed by different treatments, as indicated. As a control, an identical group of cells was kept uninfected (mock), but was incubated for 18 h in the same manner. The infection rate of the cells was *96 % at a MOI of 100.
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overnight to arrest cell in the G0 phase. The transfected ASMCs were separated into single cells and fixed. Cells were suspended with 300 ll of PBS. Ten microliters of 10 mg/ml RNase A were added to final concentration about 333 lg/ml. Cells were incubated in a 37 °C water bath for 10 min and 5 ll of 5 mg/ml PI were added to a final concentration of 80 lg/ml. Cells were stained at 37 °C protected from light for 30 min and sieved with 300 mesh screen for flow cytometry measurement. Western blotting
Cell counting kit-8 (CCK-8) assay Three thousand ASMCs at passage 3–8 were seeded onto 96-well tissue culture plates. Cells incubated in 100 ll medium were divided into the group transfected with AdGFP (optimal MOI = 100) and control in 10 wells, respectively. Airway smooth muscle cells were grown for 1, 2, and 3 days and 10 ll of the CCK-8 solution were added to each well of the plate and incubated for 2 h. Absorbance at 450 nm was measured using automatic microplate reader. Hoechst-33342 staining Ten micrograms per microliter of Hoechst stock solution was prepared and stored at -20°C and protected from light. Airway smooth muscle cells were transfected in vitro for 2, 3, 5, and 7 days and stained with 5 ll of Hoechst33342 in fresh 500 ll medium each well in dark super clean bench. Cells were incubated in dark incubator for 30–40 min. Fluorescence of each solution was measured using inverted fluorescence microscopy illuminated by blue light. Blue nuclei were visualized and took photos under microscope, low and high magnification.
Protein samples extracted from transfected cells were evenly loaded and separated using SDS-PAGE electrophoresis. Protein separated on the gel were transferred onto PVDF membrane and blocked with 5 % no fat dry milk at room temperature. The rabbit anti-human PTEN, p-Akt, p-ERK1/2, total-Akt, total-ERK1/2, Caspases-3, p21, and Cyclin D1 (Cell Signaling Technology) were separately added as primary antibodies and mouse anti human b-actin as an internal control antibody. The membrane and primary antibodies were incubated overnight under agitation at 4 °C. Subsequently, HRP conjugated goat/mouse antirabbit as secondary antibody was incubated with the membrane at room temperature for 1–1.5 h. Statistical analysis Variables were displayed with x s: Data were analyzed using SPSS software version 13.0. Comparison among groups were analyzed using One-Way ANOVA analysis. Comparison between groups were performed by LSD/ Bonferroni method. Differences were considered statistically significant at P \ 0.05.
PE-7AAD double-labeled flow cytometry analysis Results After transfection, cells were incubated at 37 °C in a humidified 5 % CO2 atmosphere for 2, 3, 5, and 7 days. Cells pellets were separated into single cells with 0.125 % trypsin. Cells were washed with ice-cold PBS. Cells were transferred to centrifuge tubes and PBS was removed. Cells were adjusted to 1 9 106/ml cell density with 19 binding buffer. Hundred microliters of cells added into EP tube were mixed well with 10 ll of PE, 380 ll of 19 binding buffer, and 10 ll of 7-AAD for flow cytometry analysis. Propidium iodide (PI) staining flow cytometry Airway smooth muscle cells in logarithmic growth phase were seeded into 75-cm2 cell culture flask. Cells were grown to 80 % confluence. The culture medium was aspirated off. Cells were incubated in serum-free DMEM
Morphology and immunohistochemistry identification of ASMCs Airway smooth muscle cells exhibited long spindle shape under inverted microscope (Fig. 1a). Airway smooth muscle cells displayed the characteristics ‘‘peak and valley’’ appearance at 80–90 % confluence (Fig. 1b). Smooth muscle phenotype marker, a-actin were identified via cell immunohistochemistry. The a-actin of ASMCs was visualized by SP method. The reaction products are shown as brownish yellow. The positive-stained ASMCs displayed brown parallel filamentary structures with many brownish granules in the cytoplasm by microscope at higher magnification (Fig. 1c). The purity of smooth muscle cells at passage 3–4 reached up to 95 %.
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Fig. 1 Morphology and immunohistochemistry identification of ASMCs. a Single ASMC had a fusiform shape (910 magnification). b The confluent ASMC growth exhibited the typical ‘‘hill and valley’’
appearance under an inverted light microscope (910 magnification). c a-SM actin in ASMCs by immunohistochemical staining (9400 magnification)
Cytotoxicity of transfected cells
by blue light (F = 1.650, 0.412, 0.085, P = 0.245, 0.674, 0.919). It demonstrated that transfection with Ad-PTEN did not cause ASMCs apoptosis (Fig. 3a–h). The occurrence of cell apoptosis among Ad-PTEN, AdGFP, and mock groups were detected by PE-7AAD doublelabeled flow cytometry at days 2, 3, 5, and 7. There was no statistical difference in the occurrence of cell apoptosis among three groups at corresponding time points (F = 1.988, 2.569, 0.127, 0.064, P = 0.191, 0.131, 0.882, 0.939). It is verified that overexpression of PTEN gene could not induce ASMCs apoptosis (Fig. 4a–f).
As shown by inverted fluorescence microscope and flow cytometry, the optimal MOI generating higher transfection effect the following day was 100:1. Cytotoxicity of cells was determined by CCK-8. The Ad-GFP groups were transfected at MOI = 100 for 1, 2, and 3 days. There was no statistical difference between the mock and Ad-GFP groups (Fig. 2, t = -0.327, 1.149, -1.732, P = 0.784, 0.270, 0.105). It indicated that transfection with Ad-GFP at a MOI of 100 had no cytotoxicity and unaffected cells growth.
PTEN overexpression inhibits cell proliferation PTEN overexpression can not induce cell apoptosis The cell apoptosis were determined with Hoechst-33342 staining at days 1, 2, 3, 5, and 7 after transfection at MOI = 100. There was no significantly difference in the occurrence of cell apoptosis among Ad-PTEN, Ad-GFP, and mock groups by fluorescence microscope illuminated
Cell proliferation were tested by CCk-8 at 1, 2, and 3 days after cells were separately transfected with Ad-GFP or AdPTEN at MOI = 100. There were statistical differences among Ad-PTEN, Ad-GFP, and mock groups with absorbance at 450 nm (F = 480.643, 106.644, 89.59, P = 0.000). The absorbance at 450 nm in the Ad-PTEN group significant lowered than in the Ad-GFP or blank control groups (P \ 0.005). But there was no statistical difference in absorbance between Ad-GFP and blank control group (Fig. 5). It suggests that transfection with Ad-PTEN at MOI = 100 inhibit cell proliferation. PTEN overexpression cause cell cycle arrested in G0/G1 phase
Fig. 2 Comparison of cell cytotoxicity between the Ad-GFP and the mock groups. Cytotoxicity of cells was determined by CCK-8. The Ad-GFP groups were transfected at MOI = 100 for 1, 2, and 3 days. There was no statistical difference between the mock and Ad-GFP groups. Data represent the mean ± SD of three independent experiments
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Using flow cytometry with PI staining, there were statistical differences in cell cycle arrested in G0/G1 phase among Ad-PTEN, Ad-GFP, and blank control cells (F = 8.333, P = 0.005), as shown in Table 1. However, no significant difference was shown in cell cycles in S or G2/M phase (F = 2.769, 2.554, P = 0.103, 0.119). Forty eight hours after transfection with adenovirus vector, cells in the Ad-PTEN group in G0/G1 phase increased, cells in S or G2/M phase decreased. It indicated that transfection with Ad-PTEN arrest cells in G0/G1 phase.
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Fig. 3 The cell apoptosis were observed with Hoechst-33342 staining at days 1, 2, 3, 5, and 7 after transfection at MOI = 100. There was no significantly difference in the occurrence of cell apoptosis among Ad-PTEN, Ad-GFP, and mock groups by fluorescence microscope illuminated by blue light. a MOI = 0 (9200 magnification); b MOI = 0 (9100 magnification); c Ad-GFP (9200 magnification); d Ad-PTEN at day 1 (9200 magnification); e Ad-PTEN at day 2 (9100 magnification); f Ad-PTEN at day 3 (9100 magnification); g Ad-PTEN at day 5 (9 200 magnification); and h Ad-PTEN at day 7 (9200 magnification). Data represent the mean ± SD of four independent experiments
PTEN overexpression inhibits activation of Akt, downregulate Cyclin D1 and upregulate p21, but have no effect on ERK1/2, Caspase-9, and Caspase-3 At the 24 h after gene transfection, expression of PTEN protein increased in the Ad-PTEN group, but no changes were shown in the Ad-GFP and blank groups. Compared with Ad-GFP and blank control group, p-Akt in the overexpressed group remarkably decreased; however, there was no statistical difference in the expression of total-Akt
among three groups (Fig. 6a). It indicated that PTEN could downregulate the level of phosphorylation, but no effect on the total-Akt levels. There was no significant difference in the expression of p-ERK1/2, total-ERK1/2, Caspase-9 , and cleaved-Caspase-3 among three groups (Fig. 6b, c). It suggested that PTEN gene had no effect on the ERK1/2, Caspase-9, and Caspase-3 levels. Compared with no change in the Ad-GFP and blank control groups, expressions of Cyclin D1 and p21 were downregulated in the AdPTEN group (Fig. 6d). It demonstrated that PTEN gene
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Fig. 4 ASMCs apoptosis tested by FCM at days 2, 3, 5, and 7. The occurrence of cell apoptosis among Ad-PTEN, Ad-GFP, and mock groups were detected by PE-7AAD double-labeled flow cytometry at days 2, 3, 5, and 7. There was no statistical difference in the occurrence of cell apoptosis among three groups at corresponding time points. a MOCK; b Ad-GFP; c Ad-PTEN at day 2; d AdPTEN at day 3; e Ad-PTEN at day 5; and f Ad-PTEN at day 7. Data represent the mean ± SD of three independent experiments
regulated cell cycle by effects on the expression of Cyclin D1 and p21.
Discussion The tumor suppressor PTEN plays a significant role in regulating cell proliferation, apoptosis. However, little is known about the role of PTEN in the proliferation and apoptosis of human ASMCs. In this study, the results have shown that overexpression of PTEN gene could not induce human ASMCs apoptosis.
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The PTEN overexpression inhibited cell proliferation and arrested cell cycle in G0/G1 phase. To further explore the molecular mechanism, expressions of p-Akt, p-ERK, cleaved-Caspase-3, and Caspase-9 were determined by the Western blots. Overexpression of PTEN gene caused downregulation of p-Akt expression, but had no effect on p-ERK, cleaved-Caspase-3, and Caspase-9 expression. Expression of Cyclin D1 decreased, but that of P21 increased in the Ad-PTEN group. Both Cyclin D1 and p21 are the major cytokine for cell regulation and control. There are possibly several mechanisms involved in the inhibition of ASMC proliferation and apoptosis by PTEN.
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Fig. 5 Effects of PTEN on the proliferation of ASMCs. The proliferation was assessed by absorbance. *P \ 0.05 vs. Ad-GFP or mock. Data represent the mean ± SD of three independent experiments Table 1 Effect of PTEN on cell cycle of ASMCs ( x s, n = 5) Group
G0/G1
S
G2/M
Ad-PTEN
90.325 ± 3.977*,#
4.968 ± 2.231
4.643 ± 2.955
Ad-GFP
78.350 ± 7.387
12.136 ± 5.617
9.458 ± 6.758
MOCK
76.035 ± 5.960
11.257 ± 6.771
12.634 ± 6.739
F
8.333
2.769
2.554
#
* P \ 0.05 vs. Ad-GFP; P \ 0.05 vs. MOCK
First, PI3K/Akt signaling has been linked to cellular proliferation in a variety of cell types [13]. In our study, we observed a significant reduction in Akt phosphorylation after Ad-PTEN infection, which is consistent with the results from previous studies. This suggests that Akt is involved in the PTEN-induced inhibition of cell proliferation and acts as an downstream effector of PTEN [14–16]. However, the ERK1/2 phosphorylation was not affected. Extracellular signal-regulated kinase 1/2 has been reported to be both a participant and non-participant in chemotaxis [17, 18], and its function varies in different cell types. Some studies showed after transfection with wild-type PTEN in colon cancer cell lines DLD-1, HT-29, and SW480, the Akt and ERK activities were downregulated [19]. However, transfection with wild-type PTEN in the normal colon fibroblast CCD-18Co could not downregulate Akt and ERK activities or induce apoptosis. In this study, the overexpression of PTEN did not inhibit the activation of ERK1/2. Our data are consistent with a certain previous studies on endothelial cell and normal colon fibroblast growth, in which PTEN overexpression did not alter the ERK1/2 activation [20]. The cellular apoptotic process is controlled by both intrinsic and extrinsic mechanisms. Activated caspase-9 can cleave and activate procaspase-3 directly, leading to a cascade of additional caspase activation and apoptosis. Akt and ERK were the primary anti-apoptotic pathway. The phosphatase and tensin homology deleted on chromosome
ten gene inhibits PI3K/Akt signal pathway by dephosphorylation of PIPS and decreased Akt activities. Subsequently, the effect of prompting cell viability was reversed to facilitate cell apoptosis. Although PTEN gene induces apoptosis of a series of tumor cell line via regulating Akt and FAk levels, and plays an important role in apoptosis of ovary cancer cells [21]. However, in our study, overexpressed PTEN gene only downregulate Akt levels, while the effect of PTEN on ASMCs is limited; it could not activate Caspase cascades to induce cell apoptosis. It might be due to different effect of PTEN gene between tumor and normal cells, or PTEN has different impact on specific signal factors in ASMCs. Another study has also demonstrated that PTEN overexpression inhibits cell proliferation, migration, and survival in rabbit VSMCs. It is likely that the inhibition of VSMC proliferation by PTEN was mediated in part by an increase in apoptosis [22]. However, other findings have indicated that exogenous PTEN suppresses cell growth and cell cycle progression without inducing cell death [23, 24]. These findings could explain the cell type and species differences in PTEN-regulated apoptosis. The discrepancies could be attributable to the differences in PTEN signaling in VSMC and other cell types. It is reported that PTEN gene in tumor cells induce cyclin-dependent kinase (CDKs) and inhibit expression of factors p21, p27, and p51. Therefore, PTEN gene can control the progress of cell cycle and regulate cell growth [25]. The phosphatase and tensin homology deleted on chromosome ten gene inhibited myocardial cell proliferation via PI3-K and Ca2?/calcineurin phosphatase signaling pathways in cardiovascular system. Overexpression of PTEN in vascular smooth muscle cell could downregulate Cyclins and CDKs, upregulate expression of CDK inhibitors p21 and p27, arrest cells in G0/G1 phase, and suppress expression of TNF-a-induced matrix metalloproteinase-9 (MMP-9) [26]. In our study, PTEN gene in ASMCs induced cell cycle arrested in the G0/G1 phase via downregulation of Cyclin D1 and upregulation of p21 expression. It was consistent with cardiovascular effect of PTEN. However, this is in discrepancy with other studies indicating that expression of PTEN gene arrested colon cancer cells in G2 phase while not in G0/G1 phase [19]. Further studies are warranted to ascertain the inconsistence whether intrinsic PTEN gene plays an important role in different cells. It is shown by other studies that, although wild-type PTEN gene transfection can inhibit cell growth and induce cell apoptosis, some cell lines only displayed cell cycle arrest, not cell apoptosis. For example, it was found in a study about MCF-7 breast cancer cells that overexpression of PTEN could induce tumor cell apoptosis, but much later than cell cycle arrest or occurred possibly only in the cells
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Fig. 6 The expression of PTEN, phospho-Akt, total-Akt, phosphor-ERK, total ERK, Caspase-9, cleaved-Caspase-3, p21, Cyclin D1, and b-actin or GAPDH in ASMCs. The cells were lysed, and the proteins were separated by SDS-PAGE and probed with antibodies against PTEN, phospho-Akt, total-Akt, phosphor-ERK, total ERK, Caspase-9, cleavedCaspase-3, p21, Cyclin D1, and b-actin or GAPDH. *P \ 0.05 vs. Ad-GFP or mock
arrested in G1 phase and cultured in serum-free culture medium [25]. However, Weng et al. [27] found that apoptosis induced by PTEN gene occurred not in the cells cycle arrested in the G1 phase. It appears to be consistent with our study showing that overexpression of PTEN gene in ASMCs only induced cell cycle arrested but no apoptosis happened. It suggests that there is no necessary link between cell cycle arrest and cell apoptosis. It is hypothesized that effect of PTEN gene on cell cycle and apoptosis regulation might vary due to different molecular backgrounds, or PTEN gene could induce only cell cycle arrest, not cell apoptosis in specific cell line. In conclusion, our study indicated that overexpression of PTEN gene did not induce apoptosis of human ASMCs cultured in vitro. However, cell proliferation was inhibited and cell cycle was arrested via downregulation of p-Akt and Cyclin D1 expression and upregulation of p21 expression. The adenovirus-mediated PTEN overexpression provides the basis of further study on effect of PTEN on airway modeling of bronchial asthma in vitro. It contributes to related study on asthmatic airway remodeling.
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Acknowledgments This study was supported by the National Natural Science Foundation of China (grant no. 30770936). We are grateful to the Department of Thoracic Surgery of Nanfang Hospital for their co-operation in obtaining the resected lung specimens, and we thank Dr Kenneth M. Yamada (National Institutes of Health, Bethesda, MD) for kindly providing the PTEN cDNA.
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