Affiliations of authors: J. O. Boyle (Laboratory of Molecular Medicine and Head and Neck Service), J. Langenfeld (Laboratory of Molecular Medicine and Thoracic Surgery Service), F. Lonardo (Laboratory of Molecular Medicine and Department of Pathology), D. Sekula (Laboratory of Molecular Medicine), V. Rusch (Laboratory of Molecular Medicine and Thoracic Surgery Service), E. Dmitrovsky (Laboratory of Molecular Medicine and Molecular Pharmacology and Therapeutics Program), Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY; P. Reczek, Bristol-Myers Squibb Pharmaceutical Research Institute, Buffalo, NY; M. I. Dawson, Retinoid Program, SRI International, Menlo Park, CA.
Correspondence to: Jay O. Boyle, M.D., Head and Neck Service,Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021 (e-mail: jboyle{at}mskcc.org).
Present address:D. Sekula, Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH.
Present address: E. Dmitrovsky, Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH.
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ABSTRACT |
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INTRODUCTION |
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We have reported that all-trans-retinoic acid (RA) can prevent the transformation of human bronchial epithelial cells in vitro(7); i.e., RA inhibited the transformation of immortalized human bronchial epithelial cells (BEAS-2B) that had been treated with tobacco-derived carcinogens. This in vitro chemopreventive activity was associated with a decline in the expression of cyclin E, the arrest of cells in G1 phase of the cell cycle, and the concomitant suppression of growth (7). A decline in the amount of cyclin D1 protein also followed treatment of these human bronchial epithelial cells with RA and was blocked by inhibition of the 26S proteasome degradation pathway (8), a finding that suggests that proteasome-dependent degradation of cyclin D1 is a mechanism used by retinoids to prevent the carcinogen-induced transformation of human bronchial epithelial cells.
In this study, we have investigated whether this is a common mechanism by studying additional types of human bronchial epithelial cells and various carotenoids and retinoids to determine whether these compounds use the degradation of cyclin D1 as a common chemoprevention mechanism.
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MATERIALS AND METHODS |
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Primary normal human bronchial epithelial cells (Clonetics, San Diego, CA) were cultured as previously described (8) in modified LHC-9 medium in the presence or absence of the indicated retinoids, carotenoids, or appropriate vehicle controls. Cells were passaged every 5-7 days and were used for experiments after two to four passages. BEAS-2B cells were derived from human bronchial epithelial cells immortalized with an adenovirus 12-simian virus 40 hybrid virus (9). BEAS-2BNNK cells are carcinogen-transformed human bronchial epithelial cells derived from BEAS-2B immortalized bronchial epithelial cells after treatment with N-nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, as described (7). These immortalized or transformed human bronchial epithelial cells were cultured in serum-free medium by established techniques (10). These cells were treated with the indicated retinoids or carotenoids in subdued light and then cultured in the dark in an incubator with humidified air at 37 °C with 5% CO2, as previously described (7-10).
Retinoid Treatments
The receptor-nonselective retinoids used were RA,
9-cis-retinoic acid (9-cis-RA), and
13-cis-RA (Sigma Chemical Co., St. Louis, MO). The
receptor-selective retinoid agonists were Am80 (selective for retinoic
acid receptor [RAR
]) (11), SR11254
(RAR
/ß) (12), SR11246 and SR11345 (retinoid X
receptor [RXR]) (13) (SRI International, Menlo Park, CA),
and BMS-189453 (RARß) (14) (Bristol-Myers Squibb, Buffalo,
NY). Stock solutions of retinoids (10-2M)
dissolved in dimethyl sulfoxide were stored in the dark at -70 °C until used.
Carotenoids
Stock solutions of the carotenoids -carotene (10-2
M) and ß-carotene (10-2 M) (Sigma
Chemical Co.) were individually dissolved in tetrahydrofuran. These
stock solutions were added to LHC-9 medium at room temperature with
vigorous stirring to achieve a final concentration of 10-5
M. The concentrations of these stock carotene solutions were
verified spectrophotometrically, and the solutions were distributed in
aliquots and stored in the dark at -70 °C
(15,16). Before use, the carotene stock solutions were diluted
to the indicated concentrations with LHC-9 medium.
Immunoblot Analysis
The human bronchial epithelial cells examined were treated with the
indicated retinoids, carotenoids, or appropriate vehicle solutions for
0-24 hours before lysis on 10-cm tissue culture plates (Falcon,
Franklin Lakes, NJ) with buffer containing protease inhibitors, as
described (7). Total cellular protein was measured by the
Bradford assay, and 100-200 µg of total cellular protein was
denatured, subjected to electrophoresis through 10% polyacrylamide
gels containing sodium dodecyl sulfate, and electroblotted to
nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). The
following primary antibodies were used: for human cyclin D1, M-20; for
RAR, C-20; for RARß, C-19; for RAR
, C-19; and for
RXR
, D-20 (all from Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). A polyclonal anti-rabbit immunoglobulin antibody was used as the
secondary antibody (Amersham Life Science Inc., Arlington Heights, IL).
Antibody was detected with the Amersham chemiluminescence assay.
To verify the specificity of each anti-retinoid receptor antibody, preincubation of the antibody with a 10-fold excess of a specific blocking peptide at room temperature for 2 hours was found to abolish the expected immunoblot signal. The blocking peptides used for each antibody (Santa Cruz Biotechnology, Inc.) were the immunogenic peptides used to produce the corresponding antibodies.
Thymidine Proliferation Assay
Growth of human bronchial epithelial cells was assessed by tritiated thymidine incorporation. Approximately 5 x 104 cells (plated per well in a six-well tissue culture plate [Falcon]) were incubated for 1 hour in tritiated thymidine (4 µCi/mL; Du Pont NEN, Boston, MA) at 37 °C, washed with phosphate-buffered saline, treated with 5% trichloroacetic acid, washed with 70% ethanol, air dried, and lysed in a solution of 10 mM NaOH and 1% sodium dodecyl sulfate. Lysates were collected, and the amount of tritiated thymidine incorporated was measured by scintillation counting. The amount of radioactivity detected (decays per minute) in each treatment sample was expressed as a percentage of the amount of the radioactivity in control samples. Data are reported as the average of six data points from three independent experiments. Error bars represent the standard deviation for each average value.
Inhibition of the Proteasome Degradation Pathway
To inhibit the 26S proteasome degradation pathway, we treated BEAS-2B immortalized human bronchial epithelial cells with calpain inhibitor I (100 µM) with or without a retinoid for 4-6 hours at 37 °C before cell lysis and immunoblot analysis of the isolated total cellular protein. No cytotoxicity was detected as a result of the calpain inhibitor I treatment under these culture conditions.
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RESULTS |
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Earlier work indicates that a link exists between cyclin D1 proteolysis
and RA-mediated prevention of carcinogen-induced transformation of
immortalized human bronchial epithelial cells (7,8). To extend
this work, we measured cyclin D1 protein by immunoblot analysis in
normal, immortalized, and transformed human bronchial epithelial cells
after treatment with RA or vehicle alone to determine whether the
observed RA-induced growth suppression parallels a decline in the
abundance of cyclin D1. In these human bronchial epithelial cells, the
amount of cyclin D1 protein declined after treatment with 2
µM RA, as shown in Fig. 2. In marked
contrast, when these human bronchial epithelial cells were treated with
2 µM ß-carotene, the amount of cyclin D1 protein
detected did not change appreciably. Similar findings were observed
after treatment with
-carotene (data not shown). Thus, the
suppression of the growth of human bronchial epithelial cells after
treatment with retinoids but not with carotenoids is tightly linked to
the expression of cyclin D1 protein.
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When BEAS-2B cells were treated with ligands specific for each type of
RARs and for RXRs, only ligands (agonists) that activated the RARß
or RXR pathway suppressed the growth of these cells (Fig.
3). When BEAS-2B cells were treated with the
RARß-specific agonist BMS-189453 (0-1.0 µM) or with
the RXR-selective agonist SR11246 (0-2.0 µM), a
dose-dependent suppression of growth was observed. When BEAS-2B cells
were treated with another RXR agonist, SR11345 (0-2 µM;
data not shown), a dose-dependent decline in growth was also
observed. When BEAS-2B cells were treated with the RAR
-specific
agonist Am80 (0-1.0 µM) or with the
RAR
/ß-selective agonist SR11254 (0-0.1 µM), cell
growth was not suppressed. When BEAS-2B immortalized human bronchial
epithelial cells were treated with the RARß (BMS-189453) and the
RXR (SR11246) agonists, additive effects on growth suppression were
observed (data not shown).
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DISCUSSION |
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Deregulated expression of cyclin D1 protein occurs in epithelial carcinogenesis [(26,27); Lonardo F, Langenfeld J, Rusch V, Dmitrovsky E, Klimstra DS: unpublished data]. Immunohistochemical findings indicate that cyclin D1 overexpression is frequent in neoplastic and carcinogen-exposed human lung epithelial cells [(27); Lonardo F, Langenfeld J, Rusch V, Dmitrovsky E, Klimstra DS: unpublished data]. Amplification of the cyclin D1 gene locus is often detected in squamous cell carcinomas of the lung or head and neck by comparative genomic hybridization or Southern blot analysis (26,28,29). It has been hypothesized (8) that cyclin D1 overexpression in human bronchial epithelial cells triggers aberrant cellular proliferation. The retinoid-mediated decrease in cyclin D1 likely restores a more normal proliferation state to these neoplastic bronchial epithelial cells.
The RA-mediated prevention of carcinogen-induced transformation of
immortalized human bronchial epithelial cells has been shown to be
linked to a post-translational regulation of G1 cyclins
(7,8). The current study extends this work by revealing that
retinoids but not carotenoids act through a common cancer
chemoprevention signal: cyclin D1 proteolysis by activation of a
proteasome-dependent degradation pathway. Growth suppression signaled
by retinoid receptor-selective agonists was also found to be associated
with cyclin D1 proteolysis by a post-translational mechanism that is
similar to the mechanism used by RA, as shown in Fig. 4. This pathway
is activated by retinoid treatment of normal, immortalized, and
transformed human bronchial epithelial cells. Similar findings were
obtained by treatment of immortalized human bronchial epithelial cells
with another proteasome inhibitor, lactacystin (8). Thus,
these findings indicate that a decline in the expression of cyclin D1
protein with concomitant growth suppression can be viewed as a retinoid
chemoprevention signal active in human bronchial epithelial cells.
Whether other candidate chemoprevention agents active in the prevention
of lung cancer use a mechanism involving the post-translational
degradation of cyclin D1 is the subject of future work.
The induction of RARß protein by RA and the observed effects of an RARß agonist in human bronchial epithelial cells are consistent with the hypothesis that RARß plays an important role in signaling a retinoid chemoprevention response in this cell context. These findings support and extend the work of others (30). Repression of RARß expression is frequently observed during epithelial cell transformation (30,31), and induction of RARß by treatment with 13-cis-RA is associated with a beneficial clinical response in oral leukoplakia, a premalignant lesion (30). Our results are consistent with the hypothesis that RARß plays a role in retinoid-induced growth suppression of human bronchial epithelial cells. These findings suggest a role for RARß agonists in the treatment of these neoplastic cells.
RXRs are known to heterodimerize with RARs and other members of the
steroid receptor superfamily (32). The growth-suppressive
effect of an RXR agonist (Fig. 3) is consistent with the hypothesis
that an RXR-dependent signal mediates the growth suppression observed
in these examined human bronchial epithelial cells. Notably, findings
depicted in Fig. 4
indicate that cyclin D1 proteolysis by a
proteasome-dependent degradation pathway follows treatment with an
RARß agonist or an RXR agonist. The signaling of this degradation
pathway by retinoid receptor-selective ligands is consistent with the
view that cyclin D1 proteolysis plays an important role in the
chemoprevention of human bronchial epithelial cells. Perhaps RXR
agonists will exhibit lung cancer prevention activity when tested in
appropriate clinical trials.
In this study, the growth-suppressive effects of RARß-selective and
RXR-selective agonists on immortalized human bronchial epithelial cells
are consistent with results obtained when RARß or RXR is
individually overexpressed in transformed human bronchial epithelial
cells (19). Growth-suppressive effects were not observed after
treatment of BEAS-2B cells with the RAR
/ß-selective agonist
(Fig. 3
) or after transfection of RAR
into transformed human
bronchial epithelial cells (19). These findings argue against
RAR
activation playing a major role in the retinoid-induced growth
suppression of these human bronchial epithelial cells.
Studies of retinoid-dependent lung cancer prevention mechanisms in in vitro models are useful to identify intermediate markers of effective retinoid chemoprevention, to highlight potential pharmacologic targets, and to select agents appropriate for testing in clinical trials. Changes in intermediate markers represent candidate surrogate end points for clinical cancer chemoprevention trials, and these may prove useful to predict beneficial clinical responses. Validated surrogate end points will enhance conduct of short-term clinical cancer chemoprevention trials by revealing early chemoprevention responses before the long-term clinical end point of reduced cancer incidence is available. This will expedite testing of candidate clinical cancer chemoprevention agents. The finding that cyclin D1 proteolysis is a common retinoid-dependent signal in normal, immortalized, and transformed human bronchial epithelial cells suggests that cyclin D1 proteolysis is a candidate intermediate end point for future retinoid-based clinical cancer chemoprevention trials.
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NOTES |
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We thank Dr. Curtis Harris, Laboratory of Human Carcinogenesis, National Cancer Institute, for the gift of the BEAS-2B cell line.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Hong WK, Lippman SM, Itri LM, Karp DD, Lee JS, Byers RM, et al. Prevention of second primary tumors with isotretinoin in squamous-cell carcinoma of the head and neck. N Engl J Med 1990;323:795-801.[Abstract]
2 Pastorino U, Infante M, Maioli M, Chiesa G, Buyse M, Firket P, et al. Adjuvant treatment of stage I lung cancer with high-dose vitamin A. J Clin Oncol 1993;11:1216-22.[Abstract]
3
Muto Y, Moriwaki H, Ninomiya M, Adachi S, Saito A, Takasaki
KT, et al. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in
patients with hepatocellular carcinoma. Hepatoma Prevention Study Group. N Engl J Med 1996;334:1561-7.
4
Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B,
Cook NR, et al. Lack of effect of long-term supplementation with beta carotene on the incidence
of malignant neoplasms and cardiovascular disease. N Engl J Med 1996;334:1145-9.
5
Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen
MR, Glass A, et al. Effects of a combination of beta carotene and vitamin A on lung cancer and
cardiovascular disease. N Engl J Med 1996;334:1150-5.
6
The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of
vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994;330:1029-35.
7 Langenfeld J, Lonardo F, Kiyokawa H, Passalaris T, Ahn MJ, Rusch V, et al. Inhibited transformation of immortalized human bronchial epithelial cells by retinoic acid is linked to cyclin E down-regulation [published erratum appears in Oncogene 1996;13:2743]. Oncogene 1996;13:1983-90.[Medline]
8
Langenfeld J, Kiyokawa H, Sekula D, Boyle J, Dmitrovsky E.
Posttranslational regulation of cyclin D1 by retinoic acid: a chemoprevention mechanism. Proc Natl Acad Sci U S A 1997;94:12070-4.
9 Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, et al. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res 1988;48:1904-9.[Abstract]
10 Lechner JF, Haugen A, McClendon IA, Pettis EW. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 1982;18:633-42.[Medline]
11 Kagechika H, Kawachi E, Hashimoto Y, Himi T, Shudo K. Retinobenzoic acids. 1. Structureactivity relationships of aromatic amides with retinoidal activity [published erratum appears in J Med Chem 1989;32:2583]. J Med Chem 1988;31:2182-92.[Medline]
12
Yu KL, Spinazze P, Ostrowski J, Currier SJ, Pack EJ, Hammer
L, et al. Retinoic acid receptor ß,-selective ligands: synthesis and biological activity of
6-substituted 2-naphthoic acid retinoids. J Med Chem 1996;39:2411-21.[Medline]
13 Dawson MI, Jong L, Hobbs PD, Cameron JF, Chao WR, Pfahl M, et al. Conformational effects on retinoid receptor selectivity. 2. Effects of retinoid bridging group on retinoid X receptor activity and selectivity. J Med Chem 1995;38:3368-83.[Medline]
14 Chen JY, Clifford J, Zusi C, Starrett J, Tortolani D, Ostrowski J, et al. Two distinct actions of retinoid-receptor ligands. Nature 1996;382:819-22.[Medline]
15 Cooney RV, Kappock TJ 4th, Pung A, Bertram JS. Solubilization, cellular uptake, and activity of ß-carotene and other carotenoids as inhibitors of neoplastic transformation in cultured cells. Methods Enzymol 1993;214:55-68.[Medline]
16
Levy J, Bosin E, Feldman B, Giat Y, Miinster A, Danilenko M,
et al. Lycopene is a more potent inhibitor of human cancer cell proliferation than either
-carotene or ß-carotene. Nutr Cancer 1995;24:257-66.[Medline]
17 Micozzi MS, Brown ED, Edwards BK, Bieri JG, Taylor PR, Khachik F, et al. Plasma carotenoid response to chronic intake of selected foods and ß-carotene supplements in men. Am J Clin Nutr 1992;55:1120-5.[Abstract]
18 Chen GQ, Shen ZX, Wu F, Han JY, Miao JM, Zhong HJ, et al. Pharmacokinetics and efficacy of low-dose all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Leukemia 1996;10:825-8.[Medline]
19 Ahn MJ, Langenfeld J, Moasser MM, Rusch V, Dmitrovsky E. Growth suppression of transformed human bronchial epithelial cells by all-trans-retinoic acid occurs through specific retinoid receptors. Oncogene 1995;11:2357-64.[Medline]
20 Roberts JM, Koff A, Polyak K, Firpo E, Collins S, Ohtsubo M, et al. Cyclins, Cdks, and cyclin kinase inhibitors. Cold Spring Harb Symp Quant Biol 1994;59:31-8.[Medline]
21 Sherr CJ. G1 phase progression: cycling on cue. Cell 1994;79:551-5.[Medline]
22 Nurse P. Ordering S phase and M phase in the cell cycle. Cell 1994;79:547-50.[Medline]
23 Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149-63.[Medline]
24
Nasmyth K. Viewpoint: putting the cell cycle in order. Science 1996;274:1643-5.
25 Resnitzky D, Hengst L, Reed SI. Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1. Mol Cell Biol 1995;15:4347-52.[Abstract]
26 Betticher DC, Heighway J, Hasleton PS, Altermatt HJ, Ryder WD, Cerny T, et al. Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer. Br J Cancer 1996;73:294-300.[Medline]
27 Betticher DC, Heighway J, Thatcher N, Hasleton PS. Abnormal expression of CCND1 and RB1 in resection margin epithelia of lung cancer patients. Br J Cancer 1997;75: 1761-8.[Medline]
28 Petersen I, Bujard M, Petersen S, Wolf G, Goeze A, Schwendel A, et al. Patterns of chromosomal imbalances in adenocarcinoma and squamous cell carcinoma of the lung. Cancer Res 1997;57:2331-5.[Abstract]
29 Bockmuhl U, Schwendel A, Dietel M, Petersen I. Distinct patterns of chromosomal alterations in high- and low-grade head and neck squamous cell carcinomas. Cancer Res 1996;56:5325-9.[Abstract]
30
Lotan R, Xu XC, Lippman SM, Ro JY, Lee JS, Lee JJ, Hong
WK. Suppression of retinoic acid receptor-ß in premalignant oral lesions and its
up-regulation by isotretinoin. N Engl J Med 1995;332:1405-10.
31 Xu XC, Sneige N, Liu X, Nandagiri R, Lee JJ, Lukmanji F, et al. Progressive decrease in nuclear retinoic acid receptor ß messenger RNA level during breast carcinogenesis. Cancer Res 1997;57:4992-6.[Abstract]
32 Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83:841-50.[Medline]
Manuscript received June 12, 1998; revised December 2, 1998; accepted December 21, 1998.
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