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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 336-344, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0021OC

Insulin-Like Growth Factor-I Inhibits Cell Growth in the A549 Non-Small Lung Cancer Cell Line

Yuzo Kodama, Robert C. Baxter and Janet L. Martin

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales, Australia

Address correspondence to: Dr. Janet Martin, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: janetlm{at}med.usyd.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factors (IGFs) are potent mitogenic and antiapoptotic factors for many cell types, including some normal and neoplastic lung cells in vitro. However, in this study we show that IGF-I, at concentrations of 10 ng/ml or greater, significantly inhibits DNA synthesis and cell proliferation in a human lung adenocarcinoma cell line, A549. Inhibition of DNA synthesis was completely reversed by an IGF-I receptor–neutralizing antibody, {alpha}IR-3, indicating that IGF-I receptor activation is involved in its inhibitory effect. Attenuation of the p44/42 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways downstream of the IGF-I receptor using the inhibitors PD98059 and LY294002, respectively, partially reversed IGF-I–induced inhibition. Acute (2–60 min) and chronic (24 h) exposure of A549 cells to 100 ng/ml IGF-I resulted in sustained phosphorylation of Akt/protein kinase B downstream of PI 3'-kinase, whereas p44/42 MAPK phosphorylation was decreased in response to chronic exposure to IGF-I. An IGF-I dose-dependent increase in the cyclin-dependent kinase inhibitor p21Cip1/WAF1 was also observed over 24 h of treatment. Collectively, these data suggest that IGF-I is growth inhibitory to A549 cells, possibly via sustained activation of the PI 3'-kinase signaling pathway, and induction of p21Cip1/WAF1.

Abbreviations: anti-insulin like growth factor receptor I antibody, {alpha}IR-3 • bovine serum albumin, BSA • cell-conditioned media, CM • 4,6-diamidino-2-phenylindole, DAPI • ethylenediaminetetraacetic acid, EDTA • epidermal growth factor, EGF • fetal calf serum, FCS • insulin-like growth factor, IGF • IGF-I receptor, IGF-IR • IGF-binding protein, IGFBP • insulin receptor substrate, IRS • [long Arg3]IGF-I, [LR3]IGF-I • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • phosphatidylinositol 3'-kinase, PI 3'-kinase • phosphatase and tensin homolog deleted on chromosome ten, PTEN • protein phosphatase 2A, PP2A • radioimmunoassay, RIA • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • serum-free medium, SFM • Tris-HCl buffered saline, TBS • transforming growth factor-{alpha}, TGF-{alpha} • 12-O-tetradecanoylphorbol-13-acetate, TPA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is one of the leading causes of cancer mortality in both men and women, and its incidence is increasing all over the world. The average 5-yr survival rate of lung cancer patients is ~ 14% (1), and this result has not improved for 30 yr. The long-term survival rate of patients with non–small cell lung carcinoma remains unsatisfactory, even when they undergo complete and potentially curative surgery. Therefore, there is an urgent need for new strategies aimed to improve lung cancer management. It is also essential to know how lung cancer cell growth differs from normal cell development and proliferation.

The insulin-like growth factors (IGFs)-I and -II are important mitogens for normal and neoplastic cell types (2, 3) and are believed to regulate cell proliferation in an autocrine/paracrine way (2–4). The mitogenic actions of IGFs are initiated by interaction with the IGF-I receptor (IGF-IR), which has high affinity for IGF-I and IGF-II, and low affinity for insulin. The cellular events that follow IGF-I binding and account for its biologic effects are still being elucidated, but signaling pathways that are activated upon IGF-I binding have been defined. IGF-IR is a receptor protein kinase expressed in a wide variety of cell types, including mesenchymal, epithelial, and hematopoietic cells. The receptor is a transmembrane heterotetramer consisting of two {alpha} subunits and two ß subunits linked by disulfide bonds (5). Ligand binding to the receptor results in receptor oligomerization, activation of tyrosine kinase, and intermolecular receptor autophosphorylation, followed by phosphorylation of members of a family of insulin receptor substrates (IRSs) and the other molecules, including Shc, Crk, and Grb2 (5). Subsequent to these initial events, two signaling pathways that mediate many effects of IGF-I are activated: the phosphatidylinositol 3'-kinase (PI 3'-kinase) and mitogen-activated protein kinase (MAPK) pathways (5). Phosphorylation of cellular substrates in these signaling pathways consequently leads to gene activation, DNA synthesis, and cell proliferation.

It has been reported that in vitro, some lung cancer cell lines express IGF mRNAs and produce IGF-I or -II, and cell growth is stimulated by IGF-I (4). In this study we unexpectedly found in A549 cells that IGF-I inhibited DNA synthesis and cell proliferation, and have investigated possible mechanisms by which this occurred.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Media for cell culture, glutamine, bovine serum albumin (BSA), bovine insulin, hydrocortisone, and epidermal growth factor (EGF) were purchased from Sigma (St. Louis, MO). Cholera enterotoxin was purchased from ICN Biomedicals Australia (Seven Hills, New South Wales, Australia). Fetal calf serum (FCS) was purchased from Trace Biosciences (North Ryde, New South Wales, Australia). Tissue culture plasticware was supplied by Nunc (Roskilde, Denmark) and Corning, Inc. (Corning, NY). Receptor grade [long Arg3]IGF-I ([LR3]IGF-I) was obtained from GroPep Pty. Ltd. (Adelaide, South Australia, Australia), and recombinant human IGF-I was donated by Genentech (South San Francisco, CA). The MAPK kinase inhibitor PD98059, the PI 3'-kinase inhibitor LY294002, and monoclonal antibody against type I IGF receptor, {alpha}IR-3, were obtained from Calbiochem-Novabiochem (Alexandria, New South Wales, Australia). Polyclonal antibodies against Thr202/Tyr204 phosphorylated and total p44/42 MAPK, and Ser473 phosphorylated and total Akt/protein kinase B (Akt) were purchased from Cell Signaling (Beverley, MA). Mouse monoclonal p21Cip1/WAF1 antibody was purchased from Transduction Laboratories (Lexington, KY). Electrophoresis reagents and protein molecular weight markers were purchased from Amrad Pharmacia Biotech (Sydney, New South Wales, Australia) and Bio-Rad Laboratories, Inc. (Richmond, CA). Hybond C Nitrocellulose membrane and Hyperfilm enhanced chemiluminescence (ECL) were purchased from Amersham Pharmacia Biotech (Bucks, UK). Nonidet P-40 was purchased from Fluka Chemical Co. (Basel, Switzerland)

Cell Culture
The human lung adenocarcinoma cell line A549 was obtained from Dr. Maria Kavillaris at the Children's Cancer Research Institute, New South Wales, Australia. A549 cells were maintained in RPMI containing 15 mM Hepes, 10% FCS, and 2 mM glutamine. MCF-10A, a phenotypically normal human breast epithelial cell line (6) which is growth-stimulated by IGF-I (7), was used for comparison with A549 cells. MCF-10A cells were the kind gift of Drs. Robert Pauley and Herbert Soule at the Karmanos Cancer Institute, Detroit, MI. MCF-10A cells were maintained in RPMI containing 15 mM Hepes, 5% horse serum, 10 µg/ml bovine insulin, 20 ng/ml EGF, 100 ng/ml cholera enterotoxin, 0.5 µg/ml hydrocortisone, and 2 mM glutamine. Both cell lines were passaged by trypsinization every 4–5 d. For stimulation experiments, cells were incubated for 48 h in serum-free RPMI medium containing 15 mM Hepes, 2 mM glutamine, and 1 g/liter BSA (SFM) before addition of test reagents in fresh SFM.

[3H]Thymidine Incorporation
For analysis of DNA synthesis, cells were dispensed into 96-well plates at a density of 2.0 x 104 cells/well. Twenty-four hours later cells were changed into SFM for 48 h before addition of treatments. Spent media were replaced with fresh SFM containing additives such as IGF-I and intracellular signaling pathway inhibitors, and incubations were continued for 20 h. [3H]Thymidine (0.5 µCi/well) was added in 50 µl of SFM for a further 4 h incubation at 37°C. Monolayers were rinsed once with ice-cold saline and fixed with 0.2 ml/well ice-cold methanol:acetic acid (3:1) at 4°C for a minimum of 2 h. Cells were solubilized in 0.5% sodium dodecyl sulfate (SDS), and 200 µl of each lysate mixed with scintillant (UltimaGold; Packard Biosciences, Groningen, The Netherlands) before counting for 1 min in a Hewlett-Packard ß counter.

Cell Proliferation Assays
Cells were plated onto six-well plates at a density of 1.0 x 105 cells/well for 24 h. Following serum starvation for 48 h, cells were treated with or without IGF-I at the concentration of 0.1, 10, or 100 ng/ml in 2 ml SFM for 3 d. Media were removed, cells were washed with phosphate-buffered saline (PBS) twice and dispersed using trypsin-EDTA. Aliquots of suspended cells were counted using a hemacytometer.

Flow Cytometric Analysis
Cells were plated at 2 x 105 per well in 12-well plates for 24 h, then incubated with fresh SFM for 48 h before treatment with the indicated concentrations of IGF-I for 24 h. Media containing detached cells were collected, and adherent cells were dispersed using trypsin-EDTA. Cell pellets were rinsed with PBS and fixed with 70% ethanol and stored at -20°C. For analysis, pelleted cells were rinsed with PBS, and suspended in 1 ml of fluorochrome solution (50 µg/ml propidium iodide, 1 mg/ml RNase A) for at least 1 h in the dark at 4°C. Cell cycle analysis was performed using a Coulter ELITE flow cytometer (Coulter, Hialeah, FL). Twenty thousand cells were analyzed for each sample, and quantitation of cell cycle distribution was performed using Multicycle software (Phoenix Flow Systems, San Diego, CA). Labeled nuclei were gated on light scatter to remove debris, and the percentage of nuclei with a sub-G1 DNA content was determined.

Apoptosis Assay
Cells were plated at 5 x 105 per well in six-well plates for 24 h and were then incubated with SFM for 48 h. Cells were treated with or without 100 ng/ml IGF-I. After 24 h, cells were rinsed with PBS, fixed in ice-cold methanol for 10 min, and then stained with 0.8 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma). The percentage of apoptotic cells was determined microscopically as cells with visible nuclear fragmentation.

Preparation of Cell Conditioned Media and Lysates for Protein Analysis
Cells were seeded onto 12-well plates at a density of 2.0 x 105 cells per well. After 24 h the spent media were replaced with SFM, then incubated for 48 h. Cells were treated with or without 0.1, 10, or 100 ng/ml IGF-I for times indicated in individual experiments. Cell-conditioned media (CM) were collected and stored at -20°C before assay. Cells were then washed once with cold PBS and immediately homogenized in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue) or PBS-TDS buffer (PBS, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 4 mM sodium pyrophosphate, 10 mM sodium fluoride and protease inhibitor cocktail tablet (Complete; Roche Molecular Biochemicals, Mannheim, Germany), sonicated for 15 s, and frozen at -80°C.

Determination of Secreted IGFBP-3
IGFBP-3 concentrations in CM were determined by radioimmunoassay (RIA) as previously described (8). Briefly, the assay was performed in a buffer containing 0.1 M sodium phosphate, pH 6.5, 0.02% sodium azide, and 0.25% bovine albumin. Incubation mixtures (500 µl total volume) consisted of appropriately diluted samples or standards (50 µl), antiserum R100 diluted 1:20,000 (100 µl), cross-linked IGFBP-3-IGF-I tracer, 10,000 cpm (100 µl), and assay buffer (250 µl), added in that order and vortex mixed. The standard range was 0.1–20 ng of pure IGFBP-3. After 16 h incubation at 22°C, 0.5 µl normal rabbit serum and 2 µl goat anti-rabbit immunoglobulin were added and, after a further 30 min incubation, 1 ml of ice-cold 6% polyethylene glycol solution (PEG 6,000) in 0.15 M sodium chloride was added, and tubes were centrifuged 20 min at 4,200 rpm in a Beckman J-6 centrifuge (Beckman, Palo Alto, CA) cooled to 2°C. Supernatants were decanted, and the radioactivity in the pellets determined in a {gamma} counter.

SDS-PAGE and Western Blotting
For ligand blotting of secreted IGFBPs, 100 µl of CM were mixed with sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue) and boiled for 5 min, fractionated by 12% SDS-PAGE under nonreducing conditions. Proteins were transferred to Hybond C nitrocellulose membrane for 2 h at 150 mA. Blots were incubated in blocking buffer (TBS; 10 mM Tris-HCl, pH 7.4, containing 9 g/liter NaCl, 10 g/liter BSA, 0.05% Nonidet P-40, and 0.2 g/liter sodium azide) at 37°C for 2 h, then probed with 125I-labeled IGF-I (1 x 106 cpm) diluted in blocking buffer, overnight at 22°C. Blots were washed in TBS containing 0.05% Nonidet P-40, before autoradiography using Hyperfilm MP for 3 d at -80°C.

For immunoblotting signaling intermediates, protein content in PBS-TDS lysates was measured by the method of Bradford using a Bio-Rad kit (Richmond, CA), mixed with SDS-PAGE sample buffer as described above. All samples were heated at 95°C for 5 min, then fractionated on 12% gels, before transfer to nitrocellulose membrane. Blots were blocked using 5% skim milk powder in TBS containing 0.1% Tween 20 (TBS-T), then probed with the phospho-Thr202/Tyr204 p44/42 antibody (1:2,000 dilution) or p21Cip1/WAF1 antibody (1:500 dilution) in the same buffer, or phospho- Ser473 Akt antibody (1:2,000 dilution) in TBS-T containing 5% BSA overnight at 4°C and protein detected using enhanced chemiluminescence (Supersignal ECL; Pierce, Rockford, VA). Blots were stripped by submersion in commercial stripping buffer reagent (Restore Western Blot Stripping Buffer; Pierce) for 20 min at 22°C, then reprobed with total p44/42 MAPK antibody (1:2,000 dilution) or Akt antibody (1:2,000 dilution) as appropriate and ECL as before. Band densities on developed films were measured using National Institutes of Health (NIH) image software (version 1.61).

Statistical Analysis
Individual experiments were conducted in triplicate or quadruplicate wells; experiments were performed at least three times unless indicated otherwise. Data were analyzed by ANOVA and Fisher's protected least significant difference test using the StatView program for Macintosh (SAS Institute, Inc., Cary, NC); differences were considered significant where P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the A549 cell line IGF-I had a biphasic action on DNA synthesis measured by thymidine incorporation, which was significantly stimulated at up to 1 ng/ml IGF-I (P < 0.005) and unexpectedly inhibited at more than 10 ng/ml (P < 0.0001; Figure 1A) . Similar results were seen in more than 10 experiments. In contrast, IGF-I stimulated thymidine incorporation 5- to 8-fold in a dose-dependent manner in MCF-10A cells (P < 0.0001; Figure 1B). This cell line was used for the purpose of comparison because it shows a more typical response to IGF-I (7). IGF-I has previously been reported to be mitogenic for A549 cells when added either immediately after the removal of serum-containing medium, or 24 h later (2, 3, 9, 10). We evaluated whether IGF-I could inhibit DNA synthesis at different time-points, 24 and 48 h after the removal of serum, to compare the effect of IGF-I in A549 cells with previous reports. As shown in Table 1, IGF-I inhibited thymidine incorporation by ~ 60% with no difference between the different incubation times.



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  		Figure 1. The effect of IGF-I and [LR3]IGF-I on DNA synthesis in A549 cells. A549 cells (A) and MCF-10A cells (B) were treated with IGF-I (open symbols) or [LR3]IGF-I (closed symbols) at the indicated concentrations for 24 h, and DNA synthesis was determined by [3H]thymidine incorporation as described in MATERIALS AND METHODS. The data are expressed as percentage of the counts incorporated in the absence of additions (SFM), and points shown are mean ± SE of combined data from six experiments for IGF-I or two experiments for [LR3]IGF-I performed in triplicate. Significant difference in DNA synthesis compared with control (SFM) are indicated as *P < 0.05, **P < 0.005, and {dagger}P < 0.0001. Statistical significance was determined by ANOVA and Fisher's PLSD test.

 

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  		TABLE 1 Effect of IGF-I on DNA synthesis in A549 cells under different growth conditions

 
A549 cells secrete various IGFBPs, which may affect responsiveness to IGFs (3, 10). We therefore evaluated whether IGFBPs were involved in the IGF-I–induced inhibition of DNA synthesis or whether the IGF-IR was involved, using the IGF-I analog [LR3]IGF-I, which can bind to the IGF-IR but has greatly reduced affinity for IGFBPs. Similarly to IGF-I, [LR3]IGF-I at 0.1 ng/ml significantly stimulated DNA synthesis (P < 0.05) and inhibited DNA synthesis at concentrations of 3 ng/ml or greater (P < 0.0001) (Figure 1A), but with 3- to 5-fold greater potency than IGF-I. This differential effect between IGF-I and [LR3]IGF-I suggests that the inhibition does not require interaction between IGF-I and IGFBPs, but that it maybe limited by endogenous IGFBPs. We examined the secretion of IGFBPs induced by IGF-I. Media conditioned by A549 cells treated with the indicated concentrations of IGF-I were analyzed by IGF ligand blot. As shown in Figure 2A , IGFBP-3 was readily detected and increased in a dose-dependent manner by IGF-I. A 30-kD IGFBP was also detected but not affected by IGF-I treatment. We confirmed a significant increase of IGFBP-3 with IGF-I treatment by RIA (Figure 2B). However, exogenous IGFBP-3 had no inhibitory effect on DNA synthesis in A549 cells (Figure 2C), suggesting that IGFBP-3 is not responsible for the IGF-I–induced suppression of DNA synthesis.



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  		Figure 2. IGF-IR–dependent mechanism of IGF-I–induced inhibition of DNA synthesis. In A and B, media conditioned for 24 h by A549 cells in the absence or presence of IGF-I at the indicated concentrations were analyzed by ligand blotting with [125I]IGF-I (A) and IGFBP-3 RIA (B), as described in MATERIALS AND METHODS. (C) A549 cells were treated for 24 h with the indicated concentrations of IGFBP-3, and DNA synthesis was determined as described in MATERIALS AND METHODS. 100% represents the counts incorporated in the absence of additions (SFM). Data shown are the mean ± SE of combined data from two experiments performed in triplicate. (D) A549 cells were pretreated for 1 h with or without anti–IGF-I receptor antibody {alpha}IR-3 (final concentration 10 µg/ml), then IGF-I was added (final concentration 100 ng/ml) for 24 h before assessment of DNA synthesis as described in MATERIALS AND METHODS. Results are expressed as percentage of the counts incorporated in the absence of additions (SFM). Points shown are mean ± SE of pooled data from two independent experiments, performed in triplicate or quadruplicate wells. In B, significant differences compared with control are indicated as *P < 0.005, **P < 0.0001; and in D, *P < 0.05 compared with untreated control, **P < 0.001 compared with IGF-I in the absence of {alpha}IR-3 (no other treatments were significantly different from control). Statistical significance was determined by ANOVA and Fisher's PLSD test.

 
To confirm involvement of the IGF-IR in the inhibitory effect of IGF-I, we evaluated whether {alpha}IR-3 could abolish the suppression of DNA synthesis induced by IGF-I. Cells were treated with 10 µg/ml {alpha}IR-3 in the presence or absence of 100 ng/ml IGF-I. IGF-I–induced suppression of DNA synthesis in A549 cells was completely reversed by {alpha}IR-3 (P < 0.001; Figure 2D), suggesting that the inhibitory effect of IGF-I at 100 ng/ml on DNA synthesis in A549 cells is mediated through the IGF-IR. Taken together, these data indicate that intracellular signaling via the IGF-IR rather than extracellular modulation by any IGFBPs may be involved in the inhibitory effect of IGF-I in A549 cells.

We then evaluated cell growth in A549 cells treated with various concentrations of IGF-I for 3 d. The accumulation of cells over this period was significantly inhibited by IGF-I at concentrations of 10 and 100 ng/ml (P < 0.001; Figure 3A) ; a similar effect was seen after 5 d treatment with the highest dose of IGF-I (data not shown). We investigated whether induction of apoptosis could contribute to the inhibition of cell proliferation. The fragmentation of DNA characteristic of apoptosis results in a hypodiploid DNA content that can be visualized as a pre-G1 peak on a DNA cell cycle histogram. Figure 3B shows the percentage of cells in the pre-G1 peak of both control and IGF-I–treated A549 cells. The pre-G1 peak was significantly decreased in a dose-dependent manner by IGF-I (P < 0.02), indicating that IGF-I, although causing a decrease in cell number, was also inhibitory to apoptosis (Figure 3C). We also analyzed changes in nuclear morphology indicative of apoptosis using the cell-permeable DNA dye DAPI and scoring for the presence of nuclear fragmentation (Figure 3D). A significant decrease in apoptosis in IGF-I–treated cells compared with controls was observed (P < 0.0001; Figure 3E), confirming the results of the flow cytometric analysis.



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  		Figure 3. IGF-I inhibition of cell proliferation but no induction of apoptosis in A549 cells. Cells were treated with or without IGF-I at the concentrations shown for three days (A) or at 100 ng/ml for 24 h (B and D) as described in MATERIALS AND METHODS. (A) Cells were counted by hemacytometer after trypsinization. Data are shown as the increase in cell number over the 3-d test period. (B) Cells were analyzed by flow cytometry, and the percentage of cells in the pre-G1 peak was determined; the data are summarized in C. (D) DAPI-stained cells were scored for nuclear fragmentation (arrows) as a morphologic marker of apoptosis, and the percentage of apoptotic cells was determined and summarized in E. In A, points shown are mean ± SE of combined data (determined in triplicate wells) from three experiments; in C, values shown are means of three independent data sets ± SE; in E, values shown are means of two data sets ± SE (A) Significant decreases in cell number compared with control (SFM) are indicated as *P < 0.001 and **P < 0.0001. (C) Significant differences are indicated as *P < 0.02 and **P < 0.001. (E) Significant differences are indicated as *P < 0.0001. Statistical significance was determined by ANOVA and Fisher's PLSD test.

 
To examine whether intracellular signaling through the IGF-IR is involved in the IGF-I inhibitory effect, we used inhibitors of signaling intermediates of two key pathways downstream of the IGF-IR, the PI 3'-kinase pathway and the p44/42 MAPK pathway. Interestingly, IGF-I–inhibited DNA synthesis in A549 cells could partly be reversed by either LY294002 (1 µM) or PD98059 (1 µM), which inhibit the PI 3'-kinase and p44/42 MAPK pathways, respectively, although the effects of these agents were modest (P < 0.05; Figure 4) . Higher concentrations of each agent were also tested, but were strongly inhibitory to basal thymidine incorporation. The partial reversal of IGF-I inhibition by these agents suggests that both MAPK and PI 3'-kinase pathways downstream of IGF-IR activation may be involved in the suppression of DNA synthesis induced by IGF-I.



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  		Figure 4. Effect of signaling pathway inhibitors on DNA synthesis in A549 cells treated with IGF-I. A549 cells were treated for 24 h with or without 100 ng/ml IGF-I in the presence or absence of either LY294002 (to block PI 3'-kinase) or PD98059 (to block p44/42 MAPK) at 1 µM concentration, and DNA synthesis was determined as described in MATERIALS AND METHODS. The data are expressed as percentage of the counts incorporated in the absence of additions (SFM). Points shown are mean ± SE of combined data (determined in triplicate wells), with the PD98059 experiment performed two times and the LY294002 experiment performed three times. *P < 0.05 and **P < 0.001 compared with 100 ng/ml IGF-I, by ANOVA and Fisher's PLSD.

 
We then evaluated intracellular signaling pathways in A549 cells in the presence or absence of IGF-I at 100 ng/ml for the indicated times (up to 60 min) to assess the effect of acute exposure to IGF-I, or at the indicated concentrations for 24 h (chronic exposure to IGF-I). In A549 cells, IGF-I significantly stimulated Akt with sustained activation up to 60 min (Figures 5A and 5C) . MAPK showed variable activation (Figure 5B), not significant as determined by quantitating signal intensity in two separate experiments (Figure 5D). In contrast to A549 cells, MCF-10A cells showed significant activation of both Akt and MAPK by IGF-I at 4 min, declining at 8 min for Akt or at 60 min for MAPK (Figures 5A–5D). Measured after 24 h exposure to IGF-I, phosphorylation of Akt remained stimulated by 100 ng/ml IGF-I in A549 cells but not in MCF-10A cells (Figures 6A and B) . In contrast, MAPK activation was significantly decreased in a dose-dependent manner by IGF-I (P < 0.005 for A549 cells, P < 0.02 for MCF-10A cells; Figures 6C and D).



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  		Figure 5. Effect of acute exposure to IGF-I on phosphorylation of Akt and p44/42 MAPK. In A and B, cells were plated at 2.0 x 105 cells/well in 12-well plates in RPMI medium with 15 mM Hepes, 2 mM glutamine, and 10% FCS or 5% horse serum. After 24 h, the spent media were changed to serum-free medium with 0.1% BSA and 2 mM glutamine and incubated for 48 h. Cells were treated with or without IGF-I at 100 ng/ml for the indicated times (up to 60 min) and homogenized as described in MATERIALS AND METHODS. Fifty microliters of each lysate was resolved by SDS-PAGE and immunoblotted with anti–phospho-Akt (Ser473) antibody, or anti-Akt antibody, and anti–phospho-p44/42 MAPK (Thr202/Tyr204) antibody, or anti-p44/42 antibody as indicated. Data are representative of two experiments. In C and D, band densities were measured by NIH image software, and the ratio of phosphorylated to total was then calculated from two experiments and expressed as a percentage of maximal ratio. Data shown are the mean ± SE of combined data from two experiments performed in duplicate for both cells. (C) *P < 0.003 and **P < 0.0001 compared with 0 min control; {dagger}P < 0.001 compared with 4 min. (D) *P < 0.0005 compared with 0 min control, by ANOVA and Fisher's PLSD.

 


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  		Figure 6. Effect of chronic exposure to IGF-I on signaling pathways. In A and B, cells were treated with or without IGF-I at the concentrations shown for 24 h and homogenized as described in MATERIALS AND METHODS. One hundred micrograms of proteins from cell extracts was resolved by SDS-PAGE and immunoblotted with antibodies against phospho-Akt antibody, or total Akt antibody, and phospho-p44/42 MAPK, or total MAPK as indicated. In C and D, band densities were measured by NIH image software, and the ratio of phosphorylated to total was then calculated and expressed as a percentage of maximal ratio. Data shown are the mean ± SE from combined data. (C) *P < 0.0001 compared with control. (D) *P < 0.02, **P < 0.005, {dagger}P < 0.0005, and {dagger}{dagger}P < 0.0001 compared with control, by ANOVA and Fisher's PLSD.

 
Because sustained activation of Akt has previously been reported to lead to induction of the cell cycle kinase inhibitor p21Cip1/WAF1 (11), we examined this in A549 cells treated with the indicated concentrations of IGF-I. As shown in Figure 7 , p21Cip1/WAF1 was increased in a dose-dependent manner by 24 h treatment with 100 ng/ml IGF-I, suggesting a possible explanation for the decrease in DNA synthesis.



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  		Figure 7. The effect of IGF-I on induction of p21Cip1/WAF1. Cells were exposed to IGF-I at the concentrations shown for 24 h, then lysated and immunoblotting with p21Cip1/WAF1 antibody. Total Akt data were used as a loading control as shown in Figure 6E, as described in MATERIALS AND METHODS. Representative data for one of three experiments with similar results are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years the IGF axis has been shown to have important influences on cancer biology, cancer risk, and carcinogenesis (12). Population studies are providing evidence for a possible relationship between circulating levels of both IGF-I and IGFBP-3 and the risk of several common cancers, including premenopausal breast cancer, colon cancer, prostate cancer, and lung cancer (13). Moreover, in vitro studies also indicate that IGF-I acts as a mitogenic factor in many normal and neoplastic cells. In this study we have described the unexpected action of IGF-I as an inhibitor of DNA synthesis and cell proliferation in the human lung adenocarcinoma cell line A549. IGFs interact with a family of soluble IGFBPs, of which six distinct members (IGFBP-1 to IGFBP-6) have been identified and cloned (14). IGFBP-3 appears to regulate cell proliferation as a negative mediator in some cancer cell lines such as breast and lung cancer either in vitro or in vivo (15, 16). The expression and the production of IGFBPs has been evaluated in many lung cancer cell lines, including A549 cells (3, 10), which secrete various IGFBPs, predominantly IGFBP-3. We have previously shown that IGFBP-3 can act as an inhibitory factor in breast cancer cells and immortalized breast epithelial cells as a result of inhibition of DNA synthesis as well as induction of apoptosis (7, 17). In the present study, IGFBP-3 was upregulated by IGF-I, an effect also observed by others in A549 cells (18). Therefore, we hypothesized that this protein could be involved in the suppression of DNA synthesis in A549 cell treated with IGF-I.

The IGF-I analog, [LR3]IGF-I, which has extremely low affinity for IGFBPs but binds to the IGF-IR, induced the suppression of DNA synthesis in A549 cells similarly to IGF-I, but with greater apparent potency. This suggests that an IGF–IGFBP interaction is not involved in the inhibition of DNA synthesis in A549 cells treated with IGF-I, and indeed endogenous IGFBPs may act to limit this effect. We also found that exogenous IGFBP-3 had no effect on DNA synthesis. It is therefore unlikely that IGFBP-3 is involved in the inhibitory action of IGF-I in A549 cells, although we cannot exclude a contribution from endogenous IGFBP-3. Because IGF-I and [LR3]IGF-I activate the IGF-IR similarly, our data are consistent with a mechanism involving signaling via the IGF-IR. This is supported by the demonstration that IGF-IR antibody, {alpha}IR3 could completely reverse the IGF-I–induced suppression of DNA synthesis in A549 cells. A recent study has shown that exposure to IGF-I can reduce the sensitivity of this cell line to IGF actions, by downregulating the IGF-IR (18). However, our results indicate that the IGF-IR was still capable of effective—albeit inhibitory—signaling after 24 h exposure to IGF-I.

In direct contrast to our observation of decreased DNA synthesis caused by IGF-I in A549 cells, the same cell line was reported to show increased thymidine uptake and cell proliferation when stimulated by IGF-I after 24 h under serum-free conditions, and this effect was abolished by {alpha}IR-3 (2, 3, 9, 10). Although most of our studies were performed after a 48-h serum-free period, we saw identical results after only 24 h. The difference between our results and those previously published cannot, therefore, be explained at present, but may result from other changes in cell culture conditions, or different subclones of A549 cells in different laboratories. IGF-I at 20 ng/ml (inhibitory in our study) has recently been reported to have a biphasic effect on colon carcinoma cell lines, causing transient growth stimulation followed by growth inhibition, believed to be mediated through upregulation of p27kip1 (19). Some other growth factors, normally regarded as stimulating cell proliferation, are known to inhibit the growth of certain cell lines. Siegfried reported that EGF was inhibitory to lung tumor cells including A549 cells (20). Interestingly, the EGF receptor ligand, transforming growth factor-{alpha} (TGF-{alpha}) stimulated rather than inhibited growth in A549 cells as well as primary lung tumor cells. The opposite response of lung cancer cells to EGF and TGF-{alpha} suggests that these two growth factors are not identical in their biologic effects even though they bind to the same receptor.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) has also been reported as another negative regulator in various cell lines including A549 cells (21, 22), although it acts as a mitogenic factor in many cell lines and activates MAPK. The duration of MAPK activity may be important in determining a cellular response; for example, prolonged activation of the MAPK pathway may play a role in cell proliferation as well as in differentiation, depending on the cell type. However, in our study acute exposure of A549 cells to IGF-I had no consistent effect on MAPK activation, and 24 h exposure actually decreased MAPK phosphorylation significantly in A549 cells, which were growth-inhibited, and even more so in MCF-10A cells, which were growth-stimulated. It is therefore unclear how regulation of MAPK phosphorylation by IGF-I relates to the inhibition of DNA synthesis in these cells. Nevertheless, the MAPK kinase inhibitor PD98059 reproducibly caused a partial reversal of the inhibitory effect, and the possibility remains that inhibition of the MAPK pathway by IGF-I in A549 cells contributed to their decreased DNA synthesis.

The PI 3'-kinase inhibitor LY294002 also partially reversed the inhibitory effect of IGF-I on thymidine incorporation. Akt phosphorylation, which is PI 3'-kinase–dependent, is reported to be constitutively activated in most non–small cell lung cancer lines, although only weakly in A549 (23). In the present study, the phosphorylation of Akt increased within 4 min of exposure to IGF-I, and remained elevated at 60 min in A549 cells, in contrast to the transient response seen in MCF-10A cells. Even after 24 h exposure to 100 ng/ml IGF-I, Akt phosphorylation remained high in A549 cells. This sustained phosphorylation could indicate that the PI 3'-kinase pathway upstream of Akt remains activated, or that the dephosphorylation of Akt itself does not occur appropriately. The phosphatidylinositol phosphatase, PTEN, acts to disrupt signaling through PI 3'-kinase, and a loss of PTEN activity would be expected to cause sustained Akt activation. However, a survey of PTEN mutations in non-small lung cancer cell lines found that the great majority—23 out of 25, including A549—lacked PTEN mutations or deletions, although 40% of small cell lung cancer lines had intragenic PTEN deletions (24). Dephosphorylation of Akt by the protein phosphatase PP2A is also believed to play a major role in regulating Akt activity (25). Although PP2A mutations are reported in lung cancer (26), the PP2A status of A549 cells has not been described. Because non-small lung cancer cell lines with constitutively active Akt/PKB are resistant to chemotherapy and irradiation (23), A549 cells with sustained active Akt/PKB caused by treatment with IGF-I might have resistance to chemotherapy and irradiation, though this remains to be proven.

Although Akt phosphorylation may be associated with growth-stimulatory signaling through PI 3'-kinase, sustained Akt upregulation has also been reported to induce the cell cycle inhibitor p21Cip1/WAF1 (11), which at high concentration binds to, and inhibits, cyclin–cyclin-dependent kinase complexes (27). Akt has recently been shown to phosphorylate p21Cip1/WAF1, resulting in enhanced protein stability (28). We found a strong upregulation of p21Cip1/WAF1 in A549 cells after 24 h exposure to 100 ng/ml IGF-I, concomitant with the sustained increase in Akt phosphorylation. Induction of p21Cip1/WAF1 by IGF-I has been described previously, in breast cancer cells, although in that situation it was paradoxically associated with increased, rather than inhibited, cell cycle activity (29). This is believed to reflect a stimulatory role of p21Cip1/WAF1 at low concentrations in the assembly of cell cycle kinase complexes (27). It is possible that this effect accounts for the slight stimulation of DNA synthesis observed at low IGF-I concentrations in A549 cells. In other situations where p21Cip1/WAF1 upregulation by growth factors has been described, the effect on cell proliferation has been inhibitory (30, 31). The increase in p21Cip1/WAF1 caused by IGF-I that we observed in A549 cells may therefore account for their inhibited DNA synthesis and proliferation.

Despite its possible role in p21Cip1/WAF1 induction in A549 cells, Akt is known as a potent cell survival factor, inhibiting the pro-apoptotic proteins Bax and Bad, as well as other important apoptosis effectors (32). Consistent with this, IGF-I was inhibitory to apoptosis in A549 cells, as judged by flow cytometric quantitation of cells with a pre-G1 DNA content and by morphologic change with DAPI staining. Nevertheless, the increase in A549 cell number over 3 d of exposure was reduced by exposure to IGF-I. This suggests that the marked IGF-I–induced blockade of cell cycle activity caused a net decrease in cell proliferation despite the concomitant inhibition of cell death.

In conclusion, our data indicate that IGF-I may be a pleiotropic effector capable of inducing mutually exclusive cellular functions (i.e., both proliferative and antiproliferative effects) under different conditions. The outcome of IGF-I stimulation may depend not only on specific phenotypic features of A549 cells, but also on variations of the microenvironment in which the IGF-I signal is generated. This concept may have important implications for understanding the clinical effects of biologic response modifiers and other therapeutic agents when used in the management of lung cancer. Insulin has been reported to inhibit cell growth in A549 cells as well as primary lung carcinoma cells, but not normal bronchial epithelial cells (20). This suggests that A549 cells may resemble primary lung cancers in their response to agents such as IGF-I. Therefore, further investigation of the mechanism of inhibition of DNA synthesis by IGF-I in this lung cancer line may shed new light on the aberrant growth regulation of lung cancer cells.


   Acknowledgments
 
The authors thank Dr. Malcolm A. King (Clinical Immunology, Royal North Shore Hospital, Sydney) for flow cytometric analysis, and Professor Norbert Berend (Department of Respiratory Medicine, Royal North Shore Hospital, Sydney) for generous advice and discussions. This work was supported in part by grant No. 107244 from the National Health and Medical Research Council, Australia (J.L.M., R.C.B.) and a scholarship from the Graduates' Association of Juntendo University School of Medicine (Tokyo, Japan) (Y.K.).

Received in original form February 12, 2002

Received in final form April 15, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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