help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Greenberg, A. K.
Right arrow Articles by Lee, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Greenberg, A. K.
Right arrow Articles by Lee, T. C.
American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 320-328, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4710

Glucocorticoids Inhibit Lung Cancer Cell Growth through Both the Extracellular Signal-Related Kinase Pathway and Cell Cycle Regulators

Alissa K. Greenberg*, Jing Hu*, Sharmila Basu, John Hay, Joan Reibman, Ting-an Yie, Kam Meng Tchou-Wong, William N. Rom and Theodore C. Lee

Departments of Medicine, Biochemistry, Pathology, and Environmental Medicine, Division of Pulmonary and Critical Care Medicine, New York University School of Medicine; and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York

Address correspondence to: Alissa K. Greenberg, M.D., Division of Pulmonary and Critical Care Medicine, Department of Medicine, New York University School of Medicine, 550 First Avenue, Rm. NB-7N24, New York, NY 10016. E-mail: alissa.greenberg{at}med.nyu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids inhibit the proliferation of various cell types, but the mechanism of this inhibition remains unclear. We investigated the effect of dexamethasone on non–small cell lung cancer cell growth and cell cycle progression. We showed that dexamethasone suppresses the proliferation of A549 and Calu-1 cells, with accumulation of cells in G1/G0 stage of the cell cycle, as determined by fluorescence-activated cell sorter analysis. Western blot analysis confirmed that this is associated with hypophosphorylation of retinoblastoma protein. Using Western blot analysis and in vitro kinase assays, we found that dexamethasone results in decreased activity of CDK2 and 4, decreased levels of cyclin D, E2F, and Myc, and increased levels of the CDK inhibitor p21Cip1. In addition, we found that dexamethasone decreases activity of extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK). The kinetics of all these changes indicate that inhibition of the ERK/MAPK pathway precedes the cell cycle effects, suggesting that regulation of this MAPK-signaling pathway may be an alternative mechanism for glucocorticoid-induced cell cycle arrest and growth inhibition.

Abbreviations: cyclin-dependent kinase, CDK • CDK inhibitors, CKI • enhanced chemiluminescence, ECL • extracellular signal-related kinases, ERK • fluorescence-activated cell sorter, FACS • glucocorticoid receptor, GR • glucocorticoid response element, GRE • mitogen-activated protein kinase, MAPK • MAPK phosphatase, MKP • phosphate-buffered saline, PBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids are regulatory molecules that control metabolism, development, inflammation, cell growth, proliferation, and differentiation (1–4). Glucocorticoids are known to exert antiproliferative effects in a number of different tissues and cell types, including those of lymphoid, fibroblastic, and epithelial origin, resulting in G1 cell cycle arrest and/or apoptosis (4–13). These agents have also demonstrated potent inhibition of carcinogenesis in the skin, forestomach, and lung of rodents (14–19). Proposed mechanisms accounting for this growth inhibition include transcriptional repression of G1 cyclins and cyclin-dependent kinases (CDKs), and/or the transcriptional activation of CDK inhibitors (CKIs) p21 and p27 (4). However, the glucocorticoid-signaling pathway leading to cell cycle arrest and growth inhibition remains unclear.

Glucocorticoids bind to the intracellular glucocorticoid receptor (GR), a ligand-activated transcription factor. When activated, the receptor translocates into the nucleus, and as a homodimer binds specific DNA sequences, known as glucocorticoid-response elements (GREs), through which it positively or negatively regulates transcription of target genes. More complex interactions of the activated GR occur in GR-responsive genes that contain promoters with interaction sites for both the GR and for a non-GR enhancer binding protein, or that lack a GR-binding sequence but to which the GR is tethered by an interacting protein.

The cell cycle is positively regulated by the action of a family of serine/threonine kinases known as CDKs. Different CDKs function at different times of the cell cycle, and are positively regulated through binding to regulatory subunits called cyclins, a process that is essential for CDK activity. Cyclin D 1 is essential for the regulation of CDKs 4 and 6 in G1 of the cell cycle, during which time the cell commits to undergoing another round of DNA replication and cell division. Two families of proteins (CDK inhibitors, CKIs) associate with cyclin/CDK complexes to inhibit their activity. The INK4 family consists of four members (pl5, pl6, pl8, and pl9), which specifically inhibit cyclin D/CDK4 or 6 activity, and prevent entry into S phase. The Cip/Kip family consists of three members (p21, p27, and p57), which can inhibit cyclin/CDK complexes in all phases of the cell cycle, but is particularly active in inhibiting cyclin E/CDK2 activity. The orderly progression through G1 phase requires temporally coordinated interactions between G1 cyclin-dependent kinases (CDK4/6 and CDK2) and the Rb protein substrate (20). The abundance of cyclin D1 appears to be rate-limiting for G1 progression in several cell types (21–24).

Several different intracellular signaling pathways affect these cell cycle regulatory proteins. Mitogen-activated protein kinases (MAPKs) are members of discrete signaling pathways that regulate cellular responses to various extracellular growth or stress stimuli. One family of MAPKs, the extracellular signal-regulated kinases (ERKs), has been extensively described as a central component of signal transduction pathways stimulated by growth-related stimuli (25). Upon phosphorylation, ERK translocates to the nucleus, where it activates a number of immediate early genes involved in proliferation, including c-fos. Sustained activation of ERK1/2 is required for cells to pass from G1 restriction point into S phase (26, 27). This occurs, at least in part, through stimulation of the cyclin D1 promoter by activated ERK1/2 (28, 29). Deregulation of the cell cycle is frequently associated with upregulation of the Ras/Raf/MEKl/ERK pathway in cancer cells. The ERK/MAP kinases are activated by dual phosphorylation on threonine and tyrosine by MAP kinase kinases MEK1/2, which in turn are activated through phosphorylation by Raf-1 (for review, see Refs. 30–32).

In this report, we examined the role of glucocorticoids in regulating lung cancer cell growth. We looked at dexamethasone's effect on cell proliferation and cell cycle progression. We then tried to elucidate the possible mechanisms, by looking at the effects of dexamethasone on expression and activity of both the cell cycle regulatory proteins, and the MAP kinase pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Treatments
The lung cancer cell lines A549 and Calu-1 were obtained from the American Type Culture Collection. The glucocorticoid receptor negative osteosarcoma cell line U2OS was the kind gift of Dr. Michael Garabedian (New York University School of Medicine, New York, NY). A549 cells were grown in Ham's F12K medium; Calu-1 and U2OS cells were grown in Dulbecco's modified Eagle's medium. All were supplemented with 10% fetal bovine serum. Dexamethasone (Sigma-Aldrich, St. Louis, MO) was dissolved in 100% ethanol and used at a final concentration of 2 x 10-7 M in culture medium. RU486 was also provided by Dr. Garabedian, and was dissolved in 100% ethanol and used at a final concentration of 2 x 10-6 M.

Cell Proliferation Assays
A549 and Calu-1 cells were plated at 5 x 104 cells onto 100-mm tissue culture dishes. Following an overnight incubation to allow cells to adhere, ethanol carrier or dexamethasone was directly added to the cell culture medium. Dexamethasone was used at a final concentration of 2 x 10-7. An equal volume of the ethanol carrier was used to treat cells as a control. Each condition was performed in triplicate. At the indicated times, cells were trypsinized, resuspended in medium, and counted using a hemacytometer. Floating cells were pelleted from the medium and resuspended with the adherent fraction of cells for cell number and viability determinations. Cell viability was determined by the trypan blue exclusion method.

Cell Cycle Analysis
Whole cell propidium iodide staining and flow cytometric analysis was performed to determine cellular DNA content. After 3 d (A549 cells) or 5 d (Calu-1 cells) of incubation with dexamethasone (2 x 10-7) or ethanol carrier, aliquots of the cells were trypsinized, washed two times in phosphate-buffered saline (PBS), resuspended in PBS with 2% fetal calf serum and 70% ethanol while vortexing, and left at 4°C overnight. Cells were then stained at room temperature for 2–3 h in 50 µg/ml propidium iodide and 100 Kunitz U/ml RNAse A in PBS. The stained cells were measured by fluorescence-activated cell sorter (FACS) analysis (Facs Scan; Becton Dickinson) to assess DNA state. For evaluation of cell cycle, standard software was used. Gates were set to differentiate between G0/G1, S-phase, and G2-M, with apoptotic cells appearing to the left of the G0/G1 peak.

Western Blot Analysis
Whole cell lysates were prepared in lysis buffer (0.5% NP-40, 10% glycerol, 50 mM Tris-HCl pH 7.5, 0.3 mM sodium orthovanadate, 100 mM NaCl, 1 mM DTT). Protein concentrations were measured using the BioRad protein determination assay. Equal amounts of protein lysates were electrophoretically separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose membranes (Schleicher and Schuell, Inc., Burlington, VT), and immunoblotted with various primary antibodies. Antibodies to phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, Raf-1, MKP-1, MKP-2, c-Fos, glucocorticoid receptor, Rb, cyclin E, CDK4, CDK6, CDK2, p21CIPl, and p27KIP1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to phospho-Raf, phospho-MEK1/2, and MEK1/2 were obtained from New England BioLabs, Inc. (Beverly, MA). Antibody to cyclin D1 was obtained from Upstate Biotechnology (Lake Placid, NY). Secondary antibodies were obtained from Amersham-Pharmacia Biotech (Piscataway, NJ). Detection was performed with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) according to the manufacturer's instructions.

In Vitro Assay Kinase Assays for ERK and G1 Kinase Activity
The p44/42 (ERK1/2) MAPK assay was performed according to the manufacturer's (New England BioLabs) instructions, except that chemiluminescent detection was performed using ECL as described above. Briefly, monoclonal antibody to phospho-p44/42 ERK/MAP kinase was used to immunoprecipitate the active kinase from cell lysates (200 µg). The immunoprecipitate was incubated for 30 min at 30°C with 2 µg of GST–Elk-1 (amino acids 307–428 of Elk-l) fusion protein in the presence of ATP (200 µM) and 50 µl 1x kinase buffer (25 mM Tris pH 7.5, 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2). The kinase reaction was terminated with sample buffer. The reaction mixture was boiled for 5 min, and equal volumes of each sample were loaded onto an SDS-polyacrylamide gel. Following electrophoretic separation and transfer onto nitrocellulose, the blots were immunoblotted with phospho–Elk-1 (Ser383) antibody (1:1,000 dilution). Detection was performed using ECL, as described above.

For the CDK2 and CDK4 kinase assays, monoclonal antibodies to CDK2 or CDK4 (Santa Cruz) were used to immunopreciptate the active kinase from cell lysates (100 µg), with protein A beads. After washing, the immunopreciptate was incubated for 30 min at 30°C with 1 µg of the appropriate substrate, in the presence of [{gamma}-32P]ATP and kinase buffer (as above). GST-Rb769–921 (Santa Cruz) was used as substrate for CDK4, and GST-histone H1 (Santa Cruz) was used for CDK2. The reaction mixture was loaded onto an SDS-polyacrylamide gel. After the gel was dried, it was exposed to the X-ray film and developed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dexamethasone Inhibits Lung Cancer Cell Proliferation
Glucocorticoids have antiproliferative effects; however, the molecular mechanisms of these effects are not yet well characterized. To determine whether lung cancer cells respond to glucocorticoids by growth inhibition and cell cycle arrest, human non–small cell lung cancer cell lines A549 (adenocarcinoma cells) and Calu-1 (derived from epidermoid lung carcinoma metastatic to the pleura) were treated with dexamethasone or ethanol vehicle for 6 d (Figure 1) . The effect of dexamethasone on cell number was not significant after 2 d of treatment. However, in the A549 cells, dexamethasone reduced cell number by 75% by Day 5. In Calu-1 cells, we found a similar though more delayed effect on cell proliferation, with significant growth suppression after 6 d of treatment. Cell viability was > 98% in all samples with each count. These observations indicate that dexamethasone exerts a marked inhibition on cell proliferation in these lung cancer cells, in agreement with glucocorticoid effects on other cell types.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 1. Dexamethasone inhibits non–small cell lung cancer cell proliferation. A549 and Calu-1 cells were seeded on Day 0 into 100-mm dishes and cultured with vehicle only (open circles) or with 2 x 10-7 M dexamethasone (closed circles). Adherent and nonadherent cells were collected on Days 2, 4, and 6, and cell counts were determined using a hemacytometer. Cell viability (> 98%) was determined by the trypan blue exclusion method. Each point represents the mean of triplicate samples with a calculated standard error of the mean. Dexamethasone inhibited cell growth of both A549 (A) and Calu-1 (B) cells.

 
To investigate further the dexamethasone-induced growth inhibition, cellular DNA content was measured by flow cytometric analysis after treatment with dexamethasone or vehicle for 3 d (A549 cells) or 5 d (Calu-1 cells) (Figure 2) . The percentage of A549 cells that were undergoing or had just completed DNA replication (S and G2/M phases) was 41% in control cells and 21% in dexamethasone-treated cells, with a concomitant increase in the percentage of G0/G1 population of cells from 59% to 79%. Similarly, the percentages of control and treated Calu-1 cells in S and G2/M phases were 66% and 47%, respectively, and were accompanied by a rise in G0/G1 cells from 34% to 53%. These findings demonstrated that dexamethasone inhibits lung cancer cell proliferation by causing an accumulation of cells with a G1 content of DNA.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. Dexamethasone induces cell cycle arrest. A549 (A, B) and Calu-1 (C, D) cells were seeded on 100-mm dishes and cultured in the presence (B, D) or absence (A, C) of 2 x 10-7 M dexamethasone. On Day 3 for A549 cells, and Day 5 for Calu-1 cells, whole cell propidium iodide staining and flow cytometric analysis was performed to determine cellular DNA content. The tall peak represents cells in G0/G1 and the crosshatched area represents cells in S phase. Dexamethasone significantly inhibited cell proliferation and caused accumulation of cells in G0/G1 in both cell lines, with a more delayed effect in Calu-1 cells.

 
Dexamethasone Inhibits Rb Phosphorylation
G1 to S cell cycle progression is mediated by the inactivation of Rb protein through phosphorylation by G1 kinases. We investigated the role of Rb and the mechanism by which dexamethasone induced a G1 accumulation of lung cancer cells. A549 and Calu-1 cells were cultured in the presence or absence of dexamethasone. Cell lysates at various time points from 1 h to 4 d were subjected to immunoblot analysis for Rb. The phosphorylation state of Rb was assessed by its mobility in SDS polyacrylamide gel electrophoresis. The hypophosphorylated inhibitory form of Rb, which migrates faster than the hyperphosphorylated inactivated form of Rb, was the predominant Rb form resulting from dexamethasone treatment (Figure 3) . The kinetics of this effect correlated with the effect on cell growth, occurring beginning at Day 1–2 in A549 cells and Day 4 in Calu-1 calls.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 3. Affect of dexamethasone on Rb phosphorylation. A549 and Calu1 cells were cultured in the absence (-) or presence (+) of 2 x 10–7 M dexamethasone for 1–5 d, and whole cell lysates were prepared. After adjusting for total protein concentration, the proteins were resolved on SDS-10% PAGE and transferred to nitrocellulose. The blots were probed with antibodies directed against Rb. The blots shown are at the 4-d time point for both cell lines. Dexamethasone treatment resulted in predominance of the hypophosphorylated form of Rb.

 
Dexamethasone Regulation of G1 Kinases
Because the activated GR is a transcription regulator, we sought to identify possible targets among the cell cycle regulatory proteins. Functional inactivation (phosphorylation) of retinoblastoma protein allows cell cycle progression from G1 to S. This inactivation requires sequential modification by at least two distinct cyclin/CDK complexes (20). Cyclin D1 working with CDK4/6 phosphorylates Rb only partially. The complete phosphorylation of Rb is achieved by cyclin E/CDK2 complexes (20). Because dexamethasone treatment resulted in accumulation of our cells in G1, we predicted that dexamethasone would block G1 kinase activity, either directly or indirectly. We measured the activity of CDK2 and 4 by an in vitro kinase assay. A549 and Calu1 cells were cultured in the presence or absence of dexamethasone for the indicated times. CDK4 and CDK2 immunoprecipitates were isolated and assayed for in vitro kinase activity, using Rb and Histone as substrates. Dexamethasone significantly blocked the activities of both CDK4 and CDK2 kinases in both cell lines, beginning at Day 1 in A549 cells and Day 3–4 in Calu-1 cells (Figure 4) .



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. G1 cyclin kinase activity is suppressed by dexamethasone. Dexamethasone suppresses CDK2 and CDK4 activity. Cells were treated with dexamethasone (2 x 10–7 M), or vehicle for the indicated times. Whole cell extracts were prepared and immunoprecipitates of CDK4 and CDK2 were isolated for in vitro kinase assays using GST-Rb769–921 (for CDK4) and GST-histone H1 (for CDK2) as substrates.

 
We hypothesized that the glucocorticoid-induced inhibition of G1 kinase activity could be a consequence of a downregulation of the regulatory or catalytic subunits of the G1 kinase complexes (i.e., cyclin D1 and CDK4/6, or cyclin E and CDK2). Therefore, we assayed level of cyclins D1 and E, and G1 CDKs in extracts of control and glucocorticoid-treated A549 or Calu-1 cells. Although the protein expression levels of cyclin E and CDKs 2, 4, and 6 did not change with dexamethasone treatment, dexamethasone downregulated the expression of cyclin D1 in both cell lines (Figure 5) . This effect was not significant until Day 1 in A549 cells and Day 3–4 in Calu-1 cells. These findings are consistent with the possibility that the G1 accumulation of glucocorticoid-treated lung cancer cells is a result of repression of cyclin D1 expression.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 5. Expression of cell cycle regulatory proteins in response to dexamethasone. A549 and Calu-1 cells were cultured in the absence (-) or presence (+) of 2 x 10-7 M dexamethasone for 1–5 d and whole cell lysates were prepared. After adjusting for total protein concentration, the proteins were resolved on SDS-10% PAGE and transferred to nitrocellulose. The blots were probed with antibodies directed against cyclin D1, cyclin E, CDK4, CDK6, CDK2, p21WAF1/CIP1, p27KIP1, c-Myc, E2F, and ERKl/2. The blots shown are at the 4-d time point for both cell lines. Dexamethasone treatment resulted in decreased cyclin D1, c-Myc, and E2F1, and increased p21 and p27. There was no change in the levels of CDK2, CDK4, CD6 or cyclin E. The ERK blot demonstrates equal protein loading.

 
Dexamethasone Effects on Other Cell Cycle Regulatory Proteins
The antiproliferative effects of glucocorticoids could be mediated by several mechanisms. Dexamethasone could not only repress the expression or activity of gene products necessary for the G1 to S phase transition, such as the cyclins and CDKs, but it could also induce the expression of growth inhibitory molecules, such as the CKIs p21Cip1 and p27Kip1. We analyzed the expression of these putative targets in response to dexamethasone treatment. Dexamethasone strongly induced the expression of p21Cip1 in both A549 and Calu-1 cell lines. P27Kip1 was induced to a lesser extent. We also observed that c-Myc and E2F levels are downregulated by dexamethasone and may represent associated mechanisms of cell cycle inhibition (Figure 5).

Dexamethasone Inhibits the ERK/MAPK Pathway
Regulation of the cell cycle normally occurs through various intracellular signaling pathways. Glucocorticoids could be exerting their growth-inhibitory effects by regulating one of the pathways upstream of the cell cycle. The MAPK pathways are critical for cell proliferation; they may also serve as potential targets for the effects of glucocorticoids. Sustained activation of ERK1/2 is required for cells to pass the G1 restriction point and enter S phase. This occurs, at least in part, through the positive regulation of cyclin D1 expression by activated ERK1/2 (28, 29).

To determine whether glucocorticoids may regulate lung cancer cell growth and cell cycle progression through this more indirect method, we decided to examine the effect of glucocorticoids on the expression of the activated (phosphorylated) forms of the MAPKs ERK, JNK, and p38 in asynchronously growing A549 and Calu-1 cells. In general, the activity of these MAPKs is regulated not by levels of protein expression, but by phosphorylation state. Cells were treated with dexamethasone or carrier (100% ethanol) for various times (1–72 h). Both cell lines displayed constitutive and persistent expression of phospho-ERKl/2 as determined with phospho-specific antibodies to ERK1/2 in immunoblot analysis. Dexamethasone suppressed the phosphorylation of ERK1/2 in both A549 and Calu-1 cells (Figure 6A) . Total ERK1/2 expression was not affected by dexamethasone treatment. In contrast, dexamethasone did not alter the phosphorylation state of JNK or p38 over this time course.




View larger version (74K):
[in this window]
[in a new window]
  		Figure 6. Dexamethasone's effects on the MAPK pathways. (A) Dexamethasone selectively inhibits ERK/MAP kinase activation. Dexamethasone causes the dephosphorylation of activated ERK in A549 and Calu-1 cells but had no effect on JNK or p38 activation. The effect on Calu-1 cells was delayed compared with A549 cells. Cells were seeded onto 100-mm dishes and treated with ethanol vehicle (-) or 2 x 10–7 M dexamethasone (+). Whole cell lysates were prepared at the indicated times following treatment. Western blot analysis was performed using antibodies directed to phospho-ERKl/2, ERK1/2, phospho-JNK, JNK, phospho-p38, and p38 as described in MATERIALS AND METHODS. (B) ERK activity is also inhibited by dexamethasone, starting at 6 h. To confirm that reduced levels of phosphorylated ERK correlated with decreased activity, and to determine the time course of this effect, we performed in vitro kinase reactions. Whole cell extracts were prepared following treatment at the indicated times and immunoprecipitated with a phospho-ERKl/2–specific monoclonal antibody. Equal amounts of the immunoprecipitates were used in kinase reaction with GST–Elk-1 as substrate. Equal amounts of the kinase reaction products were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti–phospho-Elk-l antibody. The symbols (-) and (+) indicate the absence or presence of dexamethasone treatment. Control C1 is lysate from the 72-h dexamethasone-treated cells to which 20 ng active recombinant ERK2 was added. Control C2 is 20 ng active recombinant ERK2 in lysis buffer. (C) Dexamethasone inhibits ERK and MEK, but not Raf, and may cause sustained MKP1 activation. Dexamethasone causes dephosphorylation of activated MEK1/2, but not of activated Raf-1 (rows 1–4). MKP1 levels seemed to decrease over time; this decrease was inhibited by dexamethasone. Dexamethasone had no discernable effect on MKP2 levels. Using the same lysates obtained from the experiments performed in Figures 3 and 4, Western blot analysis was performed using anti–phospho-MEK1/2, anti-MEK1/2, anti–phospho-Raf, anti–Raf-1, anti-MKP1 and anti-MKP2 antibodies.

 
Because A549 cells express oncogenic K-Ras (mutant codon 12, gly -> ser) (33) and have constitutive activation of the Ras/Raf/ERK pathway, these cells served as our model to study further dexamethasone-induced regulation of MAPK activity and its effects on cell proliferation. To confirm that the presence of phosphorylated ERK1/2 was associated with ERK activity, and to determine the kinetics of dexamethasone's effects, we performed in vitro assays of ERK activity using a GST–Elk-1 fusion protein as substrate. ERK activity in untreated A549 cells increased over 72 h (Figure 6B). Dexamethasone suppressed the ERK1/2 in vitro phosphorylation of the GST–Elk-1 fusion protein as early as 6 h after treatment. Densitometric analysis revealed that ERK activity was suppressed by > 90% at 24, 48, and 72 h of dexamethasone treatment. These observations indicate that dexamethasone inhibition of ERK activity precedes changes in cellular DNA content and cell number, and preceded inhibition of G1 kinases.

To determine whether dexamethasone directly inhibited ERK1/2 or upstream components of the ERK pathway, we examined the effect of dexamethasone on the phosphorylation of Raf-1 and MEK1/2. For both MEK1/2 and Raf-1, specific phosphorylation correlates with kinase activity. Dexamethasone (2 x 10-7 M) suppressed the phosphorylation of MEK1/2 with a time course similar to its effect on ERK1/2. Again there was no effect on total MEK 1/2 expression. In contrast, when we tested the lysates for the effect of dexamethasone on Raf-1 phosphorylation at a site (serine 259) required for binding to 14–3-3 proteins, which are critical for Raf-1 activity (34), we observed abundant expression of phospho-Raf and no change in its phosphorylation with treatment (Figure 6C), nor did dexamethasone affect the total expression of Raf-1. Our findings demonstrate that Raf is constitutively phosphorylated at this site.

ERK activity is regulated through dephosphorylation by MAPK phosphatases (MKPs). We examined whether dexamethasone increased expression of dual specificity phosphatases MKP-1 and MKP-2, which are known to inhibit ERK activity. In untreated A549 cells MKP1 protein was expressed, and levels began to decrease somewhat by 24 h. In the presence of dexamethasone, the reduction of MKP-1 expression starting at 24 h was no longer present, and levels remained high throughout the course of the experiment. The changes in MKP-1, though not striking, were reproducible, and correlated with the changes in ERK activity (Figure 6C). MKP-2 expression was abundant in untreated cells, was unchanged over time, and was not affected by dexamethasone. Thus, dexamethasone suppression of ERK activity is associated with sustained MKP-1 expression.

Taken together, our findings suggest that both MEK1/2 and ERK1/2 are targets for inactivation by dexamethasone in a pathway that bypasses Raf activation, and may involve sustained MKP1 expression.

The GR Mediates Dexamethasone's Effects on Cell Growth, ERK, and the Cell Cycle Regulatory Proteins
We determined that the GR mediates the effects of dexamethasone on the G1 kinases, ERK, and cyclin D1. We determined that GR was present in these cell lines by Western blot analysis of whole cell lysates, using antibody to the glucocorticoid receptor (Figure 7A) . The human osteosarcoma cell line U20S has no endogenous expression of the GR, and served as a negative control.



View larger version (56K):
[in this window]
[in a new window]
  		Figure 7. Dexamethasone effects are mediated by the glucocorticoid receptor. (A) The glucocorticoid receptor is abundant in both A549 and Calu-1 cells. Protein was extracted from whole cell lysates of the various cell lines; Western blot analysis was performed using antibody to the glucocorticoid receptor. The U2OS cells are derived from osteosarcoma, and lack glucocorticoid receptor. (B) Dexamethasone effects are blocked by RU486. A GR antagonist RU486 inhibited dexamethasone-induced suppression of ERK phosphorylation and activity, cyclin D1 expression, and CDK2 and 4 activities. The various assays were performed as described above. Cells were treated for 24 h with ethanol vehicle (control), dexamethasone (2 x 10–7 M), or dexamethasone (2 x 10–7 M) + RU486 (2 x 10–6 M).

 
We then asked whether RU486, which competes with dexamethasone for the ligand-binding domain of the GR, would block the various effects of dexamethasone. We treated A549 cells for 24 h with dexamethasone (2 x 10-7 M) in the absence or presence of RU486 (2 x l0-6M). As described above, for each condition, we performed in vitro kinase assays for ERK and Rb kinase activity, and measured expression of phosphorylated ERK and cyclin D1. RU486 blocked the suppressive effect of dexamethasone on ERK, CDK4, and CDK2 kinase activities. In agreement with this finding, RU486 also blocked the dephosphorylation of phospho-ERK1/2 and the suppression of cyclin D1 expression. (Figure 7B) Thus, our findings demonstrate that dexamethasone inhibition of ERK and Rb kinase activity is dependent on the glucocorticoid receptor, and is associated with repression of cyclin D1 expression, and G1 cell cycle arrest.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we set out to investigate the mechanisms of glucocorticoids' effect on the proliferation of non–small cell lung cancer cells. Glucocorticoids inhibit the mitogen-stimulated proliferation of a variety of cells, including fibroblasts (7), mast cells (8), and lymphoid cells (5). They also inhibit cell proliferation of breast (10), cervical (35), liver (11), and lymphoid neoplasias (36), and are associated with an accumulation of cells in the G1 phase of the cell cycle. The role of glucocorticoids in lung epithelial cell proliferation remains unclear. Hydrocortisone is an important growth factor when combined with insulin, EGF, and phosphoethanolamine in cultures of normal bronchial epithelial cells (37). Yet dexamethasone suppresses proliferation of cell lines derived from human lung cancers, and immortalized rat lung alveolar epithelial cells (6, 38).

We have demonstrated that dexamethasone is capable of suppressing lung cancer cell proliferation, in agreement with these earlier observations, and does so without causing cell death. We showed that cells accumulate in G0/G1 of the cell cycle, and this is associated with hypophosphorylation of Rb protein.

There may be multiple mechanisms contributing to this cell cycle inhibition and growth arrest. Glucocorticoids inhibit the expression of cyclin D1 in airway smooth muscle cells (39), and cyclin D3, CDK4/6, cyclin E, and its partner CDK2 in lymphoid cells (5, 40, 41), with a corresponding inhibition of Rb phosphorylation. Glucocorticoid inhibition of rodent fibroblast and lung alveolar epithelial cell proliferation at G1 does not involve downregulation of G1 cyclins and CDKs, but instead, induction of the cell cycle inhibitor p21Cip1 (6, 7).

We have demonstrated that in A549 and Calu-1 cells, CDK 4 and CDK2 activity is markedly decreased by dexamethasone treatment. The levels of cyclin D1, c-Myc, and E2F-1 are also downregulated by dexamethasone. In contrast, p21 and, to a lesser extent, p27 expression is stimulated. The decreased cyclin D1 and marked increase in p21 may explain the inhibition of CDK4 activity. Inhibition of CDK2 activity may reflect the increase in p27 levels and the decrease in c-Myc (which positively regulates cyclin E/CDK2 activity [42–47]). Induction of p21 transcription by dexamethasone has previously been reported (48–50). The GRE spans a C/EBP{alpha}-binding site in the p21 promoter (49). The changes in p27 and E2F-1 may be a result of altered transcription or protein stability. Dexamethasone downregulates c-Myc at the level of transcription initiation in lymphoid cells (12, 51–53). Negative regulation of cyclin D1 may be a new, alternative mechanism of dexamethasone-induced cell cycle inhibition.

In general, studies that examine glucocorticoid inhibition of cell proliferation have focused on the role of these cell cycle regulatory proteins in mediating growth inhibition (4, 6, 7, 41). We now report a possible effect of glucocorticoids on events upstream of cell cycle changes. MAPK cascades drive specific cell cycle responses to extracellular stimuli. Sustained ERK activity is a potent promoter of G1 to S phase progression. It stimulates cyclin D1 promoter activity (28) and also increases transcription of c-Fos, which results in increased levels of AP1 and cell proliferation. Dexamethasone has been shown to inhibit the UV-induced activity of JNK in HeLa cells, and consequently AP-1 function (54). However, studies on the effects of glucocorticoids on ERK activity are very limited. Swiss 3T3 fibroblasts treated with glucocorticoids for 48 h demonstrate reduced ERK activation stimulated by insulinlike growth factor 1 (55). Glucocorticoids also suppress antigen-induced ERK activation in mast cells (8).

We found that dexamethasone selectively inhibited the activity of ERK, but not JNK or P38 in these two non–small cell lung cancer cell lines. And we have localized this inhibition to the level of MEK and ERK. Dephosphorylation and phosphorylation both control activity of both ERK and MEK. Important phosphatases that regulate ERK1/2 include MKP-1 and MKP-2. We demonstrated that dexamethasone may block the downregulation of MKP-1 in A549 cells. The mechanism by which MKP-1 expression is sustained in the presence of dexamethasone is unclear. Dexamethasone may increase MKP-1 protein stability. MKP-1 is targeted for degradation by the ubiquitin-directed proteasome complex (56). The activated GR may block steps along the ubiquitin-mediated degradation pathway. Transcriptional mechanisms by activated GR could also be involved. Were this to be a mechanism, it would likely be indirect because the effect on MKP-1 protein expression was late (12–24 h). It is likely that the inactivation of the ERK/MAPK pathway by dexamethasone may involve activation of multiple phosphatases that act downstream of Raf.

Suppression of ERK activity preceded the changes in cell cycle distribution and cyclin D1 expression. Thus, the kinetics of dexamethasone suppression of ERK activity suggest that ERK is upstream of G1 kinase regulation. There are also many possible mechanisms by which glucocorticoid-induced inhibition of the ERK/MAP kinase pathway can cause growth inhibition and cell cycle arrest. ERK inhibition is known to result in repression of cyclin D1 and c-Fos expression. Our observation that dexamethasone suppressed the expression of cyclin D1 suggested the possibility that dexamethasone might regulate cyclin D1 expression by inhibiting ERK activity. It is also possible ERK may have a more direct effect on the cell cycle by phosphorylating and thus inactivating Rb protein and/or E2F and inhibiting cell cycle progression. It has been shown that both JNK and p38 can have such a direct effect on E2F activity (57).

Our findings suggest that dexamethasone suppresses lung cancer cell proliferation through at least two signaling pathways that are dependent on the GR and involve effects on both the ERK/MAPK pathway and the cell cycle regulators. Our data strongly suggest a direct interaction between the ERK pathway and cell cycle regulators.


   Acknowledgments
 
The authors thank M. Garabedian for the generous gift of RU486 and helpful discussions. This work was supported by research grants from the Stony Wold-Herbert Foundation, the American Lung Association, and the National Institutes of Health (M0100096).


   Footnotes
 
* These authors contributed equally to the paper. Back

Received in original form August 27, 2001

Received in final form March 15, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yamamoto, K. R. 1985. Steroid receptor regulated transcription of specific genes and gene networks. Annu. Rev. Genet. 19:209–252.[Medline]
  2. Cole, T. J., J. A. Blendy, A. P. Monaghan, K. Krieglstein, W. Schmid, A. Aguzzi, G. Fantuzzi, E. Hummler, K. Unsicker, and G. Schutz. 1995. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev. 9:1608–1621.[Abstract/Free Full Text]
  3. Barnes, P. J. 1998. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin. Sci. (Colch.) 94:557–572.[Medline]
  4. Rogatsky, I., J. M. Trowbridge, and M. J. Garabedian. 1997. Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms. Mol. Cell. Biol. 17:3181–3193.[Abstract]
  5. Baghdassarian, N., R. Catallo, M. A. Mahly, P. Ffrench, F. Chizat, P. A. Bryon, and M. Ffrench. 1998. Glucocorticoids induce G1 as well as S-phase lengthening in normal human stimulated lymphocytes: differential effects on cell cycle regulatory proteins. Exp. Cell Res. 240:263–273.[Medline]
  6. Corroyer, S., E. Nabeyrat, and A. Clement. 1997. Involvement of the cell cycle inhibitor CIP1/WAF1 in lung alveolar epithelial cell growth arrest induced by glucocorticoids. Endocrinology 138:3677–3685.[Abstract/Free Full Text]
  7. Ramalingam, A., A. Hirai, and E. A. Thompson. 1997. Glucocorticoid inhibition of fibroblast proliferation and regulation of the cyclin kinase inhibitor p21Cip1. Mol. Endocrinol. 11:577–586.[Abstract/Free Full Text]
  8. Rider, L. G., N. Hirasawa, F. Santini, and M. A. Beaven. 1996. Activation of the mitogen-activated protein kinase cascade is suppressed by low concentrations of dexamethasone in mast cells. J. Immunol. 157:2374–2380.[Abstract]
  9. Ramdas, J., W. Liu, and J. M. Harmon. 1999. Glucocorticoid-induced cell death requires autoinduction of glucocorticoid receptor expression in human leukemic T cells. Cancer Res. 59:1378–1385.[Abstract/Free Full Text]
  10. Goya, L., A. C. Maiyar, Y. Ge, and G. L. Firestone. 1993. Glucocorticoids induce a G1/G0 cell cycle arrest of Con8 rat mammary tumor cells that is synchronously reversed by steroid withdrawal or addition of transforming growth factor-alpha. Mol. Endocrinol. 7:1121–1132.[Abstract]
  11. Sanchez, I., L. Goya, A. K. Vallerga, and G. L. Firestone. 1993. Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing an early G1 block in cell cycle progression. Cell Growth Differ. 4:215–225.[Abstract]
  12. Thompson, E. B., R. Thulasi, M. F. Saeed, and B. H. Johnson. 1995. Glucocorticoid antagonist RU 486 reverses agonist-induced apoptosis and c-myc repression in human leukemic CEM-C7 cells. Ann. N.Y. Acad. Sci. 761:261–275.[Abstract]
  13. Rogatsky, I., A. B. Hittelman, D. Pearce, and M. J. Garabedian. 1999. Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of glucocorticoids. Mol. Cell. Biol. 19: 5036–5049.[Abstract/Free Full Text]
  14. Sattenberg, L. W., T. S. Wiedmann, R. D. Estensen, C. L. Zimmerman, V. E. Steele, and G. J. Kelloff. 1997. Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice. Cancer Res. 57:5489–5492.[Abstract/Free Full Text]
  15. Belman, S., and W. Troll. 1972. The inhibition of croton oil-promoted mouse skin tumorigenesis by steroid hormones. Cancer Res. 32:450–454.[Abstract/Free Full Text]
  16. Estensen, R. D., and L. W. Wattenberg. 1993. Studies of chemopreventive effects of myo-inositol on benzo[a]pyrene- induced neoplasia of the lung and forestomach of female A/J mice. Carcinogenesis 14:1975–1977.[Abstract/Free Full Text]
  17. Wattenberg, L. W., and R. D. Estensen. 1996. Chemopreventive effects of myo-inositol and dexamethasone on benzo[a]pyrene and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone–induced pulmonary carcinogenesis in female A/J mice. Cancer Res. 56:5132–5135.[Abstract/Free Full Text]
  18. Wattenberg, L. W., and R. D. Estensen. 1997. Studies of chemopreventive effects of budenoside on benzo[a]pyrene-induced neoplasia of the lung of female A/J mice. Carcinogenesis 18:2015–2017.[Abstract/Free Full Text]
  19. Wattenberg, L. W., T. S. Wiedmann, R. D. Estensen, C. L. Zimmerman, A. R. Galbraith, V. E. Steele, and G. J. Kelloff. 2000. Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol. Carcinogenesis 21:179–182.[Abstract/Free Full Text]
  20. Lundberg, A. S., and R. A. Weinberg. 1998. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell. Biol. 18:753–761.[Abstract/Free Full Text]
  21. Baldin, V., J. Lukas, M. J. Marcote, M. Pagano, and G. Draetta. 1993. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 7:812–821.[Abstract/Free Full Text]
  22. Musgrove, E. A., C. S. Lee, M. F. Buckley, and R. L. Sutherland. 1994. Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle. Proc. Natl. Acad. Sci. USA 91:8022–8026.[Abstract/Free Full Text]
  23. Quelle, D. E., R. A. Ashmun, S. A. Shurtleff, J. Y. Kato, D. Bar-Sagi, M. F. Roussel, and C. J. Sherr. 1993. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev. 7:1559–1571.[Abstract/Free Full Text]
  24. Resnitzky, D., M. Gossen, H. Bujard, and S. I. Reed. 1994. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol. Cell. Biol. 14:1669–1679.[Abstract/Free Full Text]
  25. English, J., G. Pearson, J. Wilsbacher, J. Swantek, M. Karandikar, S. Xu, and M. H. Cobb. 1999. New insights into the control of MAP kinase pathways. Exp. Cell Res. 253:255–270.[Medline]
  26. Brondello, J. M., F. R. McKenzie, H. Sun, N. K. Tonks, and J. Pouyssegur. 1995. Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibroblasts. Oncogene 10:1895–1904.[Medline]
  27. Pages, G., P. Lenormand, G. L'Allemain, J. C. Chambard, S. Meloche, and J. Pouyssegur. 1993. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc. Natl. Acad. Sci. USA 90:8319–8323.[Abstract/Free Full Text]
  28. Lavoie, J. N., G. L'Allemain, A. Brunet, R. Muller, and J. Pouyssegur. 1996. Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271:20608–20616.[Abstract/Free Full Text]
  29. Weber, J. D., D. M. Raben, P. J. Phillips, and J. J. Baldassare. 1997. Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem. J. 326:61–68.
  30. Karin, M., and T. Hunter. 1995. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr. Biol. 5:747–757.[Medline]
  31. Cahill, M. A., R. Janknecht, and A. Nordheim. 1996. Signalling pathways: jack of all cascades. Curr. Biol. 6:16–19.[Medline]
  32. Lewis, T. S., P. S. Shapiro, and N. G. Ahn. 1998. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74:49–139.[Medline]
  33. Valenzuela, D. M., and J. Groffen. 1986. Four human carcinoma cell lines with novel mutations in position 12 of c-K-ras oncogene. Nucleic Acids Res. 14:843–852.[Abstract/Free Full Text]
  34. Tzivion, G., Z. Luo, and J. Avruch. 1998. A dimeric 14–3-3 protein is an essential cofactor for Raf kinase activity. Nature 394:88–92.[Medline]
  35. Samuelsson, M. K., A. Pazirandeh, B. Davani, and S. Okret. 1999. p57Kip2, a glucocorticoid-induced inhibitor of cell cycle progression in HeLa cells. Mol. Endocrinol. 13:1811–1822.[Abstract/Free Full Text]
  36. Yamamoto, K. R., M. R. Stampfer, and G. M. Tomkins. 1974. Receptors from glucocorticoid-sensitive lymphoma cells and two clases of insensitive clones: physical and DNA-binding properties. Proc. Natl. Acad. Sci. USA 71:3901–3905.[Abstract/Free Full Text]
  37. Lechner, J. F., A. Haugen, I. A. McClendon, and E. W. Pettis. 1982. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642.[Medline]
  38. Hofmann, J., U. Kaiser, M. Maasberg, and K. Havemann. 1995. Glucocorticoid receptors and growth inhibitory effects of dexamethasone in human lung cancer cell lines. Eur. J. Cancer 31A:2053–2058.
  39. Fernandes, D., E. Guida, V. Koutsoubos, T. Harris, P. Vadiveloo, J. W. Wilson, and A. G. Stewart. 1999. Glucocorticoids inhibit proliferation, cyclin D1 expression, and retinoblastoma protein phosphorylation, but not activity of the extracellular-regulated kinases in human cultured airway smooth muscle. Am. J. Respir. Cell Mol. Biol. 21:77–88.[Abstract/Free Full Text]
  40. Reisman, D., and E. A. Thompson. 1995. Glucocorticoid regulation of cyclin D3 gene transcription and mRNA stability in lymphoid cells. Mol. Endocrinol. 9:1500–1509.[Abstract]
  41. Rhee, K., D. Reisman, W. Bresnahan, and E. A. Thompson. 1995. Glucocorticoid regulation of G1 cyclin-dependent kinase genes in lymphoid cells. Cell Growth Differ. 6:691–698.[Abstract]
  42. Alevizopoulos, K., J. Vlach, S. Hennecke, and B. Amati. 1997. Cyclin E and c-Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins. EMBO J. 16:5322–5333.[Medline]
  43. Muller, D., C. Bouchard, B. Rudolph, P. Steiner, I. Stuckmann, R. Saffrich, W. Ansorge, W. Huttner, and M. Eilers. 1997. Cdk2-dependent phosphorylation of p27 facilitates its Myc-induced release from cyclin E/cdk2 complexes. Oncogene 15:2561–2576.[Medline]
  44. Perez-Roger, I., D. L. Solomon, A. Sewing, and H. Land. 1997. Myc activation of cyclin E/Cdk2 kinase involves induction of cyclin E gene transcription and inhibition of p27(Kip1) binding to newly formed complexes. Oncogene 14:2373–2381.[Medline]
  45. Pusch, O., G. Bernaschek, M. Eilers, and M. Hengstschlager. 1997. Activation of c-Myc uncouples DNA replication from activation of G1-cyclin–dependent kinases. Oncogene 15:649–656.[Medline]
  46. Steiner, P., A. Philipp, J. Lukas, D. Godden-Kent, M. Pagano, S. Mittnacht, J. Bartek, and M. Eilers. 1995. Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes. EMBO J. 14:4814–4826.[Medline]
  47. Steiner, P., B. Rudolph, D. Muller, and M. Eilers. 1996. The functions of Myc in cell cycle progression and apoptosis. Prog. Cell Cycle Res. 2:73–82.[Medline]
  48. Cha, H. H., E. J. Cram, E. C. Wang, A. J. Huang, H. G. Kasler, and G. L. Firestone. 1998. Glucocorticoids stimulate p21 gene expression by targeting multiple transcriptional elements within a steroid responsive region of the p21waf1/cip1 promoter in rat hepatoma cells. J. Biol. Chem. 273:1998–2007.[Abstract/Free Full Text]
  49. Cram, E. J., R. A. Ramos, E. C. Wang, H. H. Cha, Y. Nishio, and G. L. Firestone. 1998. Role of the CCAAT/enhancer binding protein-alpha transcription factor in the glucocorticoid stimulation of p21waf1/cip1 gene promoter activity in growth-arrested rat hepatoma cells. J. Biol. Chem. 273: 2008–2014.[Abstract/Free Full Text]
  50. Park, J. H., E. J. Oh, Y. H. Choi, C. D. Kang, H. S. Kang, D. K. Kim, K. I. Kang, and M. A. Yoo. 2001. Synergistic effects of dexamethasone and genistein on the expression of Cdk inhibitor p21WAF1/CIP1 in human hepatocellular and colorectal carcinoma cells. Int. J. Oncol. 18:997–1002.[Medline]
  51. Forsthoefel, A. M., and E. A. Thompson. 1987. Glucocorticoid regulation of transcription of the c-myc cellular protooncogene in P1798 cells. Mol. Endocrinol. 1:899–907.[Abstract]
  52. Ma, T., P. B. Mahajan, and E. A. Thompson. 1992. Glucocorticoid regulation of c-myc promoter utilization in P1798 T- lymphoma cells. Mol. Endocrinol. 6:960–968.[Abstract]
  53. Thulasi, R., D. V. Harbour, and E. B. Thompson. 1993. Suppression of c-myc is a critical step in glucocorticoid-induced human leukemic cell lysis. J. Biol. Chem. 268:18306–18312.[Abstract/Free Full Text]
  54. Caelles, C., J. M. Gonzalez-Sancho, and A. Munoz. 1997. Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway. Genes Dev. 11:3351–3364.[Abstract/Free Full Text]
  55. Hansson, A., K. Hehenberger, and M. Thoren. 1996. Long-term treatment of Swiss 3T3 fibroblasts with dexamethasone attenuates MAP kinase activation induced by insulin-like growth factor-I (IGF-I). Cell Biochem. Funct. 14:121–129.[Medline]
  56. Brondello, J. M., J. Pouyssegur, and F. R. McKenzie. 1999. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 286:2514–2517.[Abstract/Free Full Text]
  57. Wang, S., N. Nath, A. Minden, and S. Chellappan. 1999. Regulation of Rb and E2F by signal transduction cascades: divergent effects of JNK1 and p38 kinases. EMBO J. 18:1559–1570.[Medline]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
A. D. Bird, K. H. Tan, P. F. Olsson, M. Zieba, S. J. Flecknoe, D. R. Liddicoat, R. Mollard, S. B. Hooper, and T. J. Cole
Identification of glucocorticoid-regulated genes that control cell proliferation during murine respiratory development
J. Physiol., November 15, 2007; 585(1): 187 - 201.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
R. M. van den Berg, H. van Tinteren, N. van Zandwijk, C. Visser, A. Pasic, C. Kooi, T. G. Sutedja, P. Baas, K. Grunberg, W. J. Mooi, et al.
The Influence of Fluticasone Inhalation on Markers of Carcinogenesis in Bronchial Epithelium
Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 1061 - 1065.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
T. Parimon, J. W. Chien, C. L. Bryson, M. B. McDonell, E. M. Udris, and D. H. Au
Inhaled Corticosteroids and Risk of Lung Cancer among Patients with Chronic Obstructive Pulmonary Disease
Am. J. Respir. Crit. Care Med., April 1, 2007; 175(7): 712 - 719.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Chattopadhyay, E. Tracy, P. Liang, O. Robledo, S. Rose-John, and H. Baumann
Interleukin-31 and Oncostatin-M Mediate Distinct Signaling Reactions and Response Patterns in Lung Epithelial Cells
J. Biol. Chem., February 2, 2007; 282(5): 3014 - 3026.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Nigawara, Y. Iwasaki, M. Asai, M. Yoshida, M. Kambayashi, H. Sashinami, K. Hashimoto, and T. Suda
Inhibition of 11{beta}-Hydroxysteroid Dehydrogenase Eliminates Impaired Glucocorticoid Suppression and Induces Apoptosis in Corticotroph Tumor Cells
Endocrinology, February 1, 2006; 147(2): 769 - 772.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
H. Igarashi, K. L. Medina, T. Yokota, M. I. D. Rossi, N. Sakaguchi, P. C. Comp, and P. W. Kincade
Early lymphoid progenitors in mouse and man are highly sensitive to glucocorticoids
Int. Immunol., May 1, 2005; 17(5): 501 - 511.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
P. E. Gallagher and E.A. Tallant
Inhibition of human lung cancer cell growth by angiotensin-(1-7)
Carcinogenesis, November 1, 2004; 25(11): 2045 - 2052.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. Lam, J. C. leRiche, A. McWilliams, C. MacAulay, Y. Dyachkova, E. Szabo, J. Mayo, R. Schellenberg, A. Coldman, E. Hawk, et al.
A Randomized Phase IIb Trial of Pulmicort Turbuhaler (Budesonide) in People with Dysplasia of the Bronchial Epithelium
Clin. Cancer Res., October 1, 2004; 10(19): 6502 - 6511.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
J. Tamaoki, E. Tagaya, K. Kawatani, J. Nakata, Y. Endo, and A. Nagai
Airway Mucosal Thickening and Bronchial Hyperresponsiveness Induced by Inhaled {beta}2-Agonist in Mice
Chest, July 1, 2004; 126(1): 205 - 212.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Wang, Z. Zhang, E. Kastens, R. A. Lubet, and M. You
Mice with Alterations in Both p53 and Ink4a/Arf Display a Striking Increase in Lung Tumor Multiplicity and Progression: Differential Chemopreventive Effect of Budesonide in Wild-type and Mutant A/J Mice
Cancer Res., August 1, 2003; 63(15): 4389 - 4395.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Greenberg, A. K.
Right arrow Articles by Lee, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Greenberg, A. K.
Right arrow Articles by Lee, T. C.

HOME HELP