© 1999 by Oxford University Press
Journal of the National Cancer Institute, Vol. 91, No. 4, 373-379,
February 17, 1999
© 1999 Oxford University Press
REPORTS |
Cyclin D1 Proteolysis: a Retinoid Chemoprevention Signal in Normal, Immortalized, and Transformed Human Bronchial Epithelial Cells
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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BACKGROUND: Retinoids (derivatives of vitamin A) are reported to reduce the occurrence of some second primary cancers, including aerodigestive tract tumors. In contrast, ß-carotene does not reduce the occurrence of primary aerodigestive tract cancers. Mechanisms explaining these effective retinoid and ineffective carotenoid chemoprevention results are poorly defined. Recently, the all-trans-retinoic acid (RA)-induced proteolysis of cyclin D1 that leads to the arrest of cells in G1 phase of the cell cycle was described in human bronchial epithelial cells and is a promising candidate for such a mechanism. In this study, we have investigated this proteolysis as a common signal used by carotenoids or receptor-selective and receptor-nonselective retinoids. METHODS: We treated cultured normal human bronchial epithelial cells, immortalized human bronchial epithelial cells (BEAS-2B), and transformed human bronchial epithelial cells (BEAS-2BNNK) with receptor-selective or receptor-nonselective retinoids or with carotenoids and studied the effects on cell proliferation by means of tritiated thymidine incorporation and on cyclin D1 expression by means of immunoblot analysis. We also examined whether calpain inhibitor I, an inhibitor of the 26S proteasome degradation pathway, affected the decline (i.e., proteolysis) of cyclin D1. RESULTS: Receptor-nonselective retinoids were superior to the carotenoids studied in mediating the decline in cyclin D1 expression and in suppressing the growth of bronchial epithelial cells. Retinoids that activated retinoic acid receptor ß or retinoid X receptor pathways preferentially led to a decrease in the amount of cyclin D1 protein and a corresponding decline in growth. The retinoid-mediated degradation of cyclin D1 was blocked by cotreatment with calpain inhibitor I. CONCLUSIONS: Retinoid-dependent cyclin D1 proteolysis is a common chemoprevention signal in normal and neoplastic human bronchial epithelial cells. In contrast, carotenoids did not affect cyclin D1 expression. Thus, the degradation of cyclin D1 is a candidate intermediate marker for effective retinoid-mediated cancer chemoprevention in the aerodigestive tract.
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INTRODUCTION |
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Lung cancer is the leading cause of cancer mortality for men and women in the United States. Because curative therapy for disseminated non-small-cell lung cancer does not yet exist, effective chemoprevention of lung cancer in patients with prior tobacco exposure would have a major impact on reducing lung cancer mortality. Retinoids, natural and synthetic derivatives of vitamin A, have in vitro and in vivo chemoprevention activities. Clinical trials have shown that retinoids are active in treatment of some second cancers. Treatment with 13-cis-retinoic acid (13-cis-RA) has reduced second aerodigestive tract cancers in patients with previously resected head and neck cancers (1). Treatment with retinyl palmitate has reduced second primary lung cancers in patients who have had completely resected lung cancers (2). An acyclic retinoid, polyprenoic acid, inhibited development of second hepatocellular carcinomas in patients with resected or ablated primary hepatocellular carcinomas (3). The mechanisms used by retinoids to produce these beneficial cancer chemoprevention results are poorly defined, and vitamin A-associated toxic effects have limited chronic retinoid treatments of individuals at high risk of developing cancer. The desire to reduce retinoid toxicity led to large cancer chemoprevention trials of ß-carotene, a related compound that is clinically well tolerated; unfortunately, these trials did not result in the prevention of lung cancer in high-risk individuals (4-6). These findings underscore the potential value of in vitro carcinogenesis studies in model systems that can be used to determine cancer prevention mechanisms, to identify intermediate markers, and to select appropriate agents for testing in clinical cancer chemoprevention trials.
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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human Bronchial Epithelial Cells
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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Compared with a treatment with the vehicle control, a 24-hour treatment of BEAS-2B immortalized human bronchial epithelial cells with 4 µM RA, 4 µM 13-cis-RA, or 4 µM 9-cis-RA resulted in appreciable growth suppression, as measured by tritiated thymidine incorporation (Fig. 1, A).
Treatment of BEAS-2B cells with -carotene or
ß-carotene at concentrations up to 8 µM did not
suppress bronchial epithelial cell growth (Fig. 1, B). These
concentrations of carotenoids and retinoids in plasma are clinically
achievable levels for each agent (17,18). If the carotenoid
treatment was examined for up to 72 hours, no effect on BEAS-2B cell
growth was observed (data not shown).
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Treatment with retinoids but not with carotenoids suppressed the growth of BEAS-2B cells, normal human bronchial epithelial cells in short-term cultures, and carcinogen-transformed BEAS-2BNNK cells (Fig. 1
, C). These normal, immortalized, and transformed human bronchial
epithelial cells represent in vitro models for lung tissue of
carcinogen-exposed individuals at risk for developing primary or
secondary aerodigestive tract cancers. These models could be used for
the preliminary testing of clinical cancer chemoprevention agents.
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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Biologic effects of retinoids are mediated through the nuclear retinoid receptors RARs and RXRs. Expression of specific retinoid receptors is regulated by RA (19) at the level of messenger RNA in transformed human bronchial epithelial cells, and specific retinoid receptors are involved in regulation of basal cell growth. To determine which of these receptor proteins are involved in the retinoid-mediated decrease in the amount of cyclin D1 and resulting growth suppression in human bronchial epithelial cells, we assessed the effect of RA on the basal expression of retinoid receptor proteins by immunoblot analysis. Treatment with 2 µM RA led to a decrease in the expression of RAR
, RAR, and RXR protein, as shown in Fig. 2, C.
In contrast, the expression of RARß protein was induced after
treatment with RA. Relative to basal expression, both RAR1 and
RAR2 isoforms were repressed by RA treatment as shown in Fig. 2,
C. Basal expression of RAR protein was quite low in BEAS-2B cells
and decreased further 12 and 24 hours after treatment with RA (Fig. 2,
C; data not shown). Expression of RXR protein was also repressed
by treatment with RA (Fig. 2, C; data not shown). The induction of
RARß protein by RA was undetected at 3 hours, was maximal from 6
hours to 12 hours, and then decreased from 24 hours to 48 hours. The
specificity of the anti-retinoid receptor antibodies was confirmed
through blocking experiments in which receptor-specific peptides were
incubated with their corresponding antibodies before immunoblot
analysis. In resulting immunoblots, the specific retinoid receptor was
not detected (data not shown), thus demonstrating the specificity of
the various retinoid receptor antibodies.
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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After immortalized human bronchial epithelial (BEAS-2B) cells were treated with agonists selective for RARß or for RXRs, the abundance of cyclin D1 protein was assessed by immunoblot analysis to determine whether the growth suppression induced by each of these ligands was linked to a decline in the level of cyclin D1. Each agonist tested (RA, the RXR-selective agonist SR11246, and the RARß-selective agonist BMS-189453) at growth-suppressing doses caused a dose-dependent decline in the abundance of cyclin D1 protein (Fig. 4).
This
confirmed that a tight link exists between the growth suppression
signaled by these selective retinoid receptor agonists and the level of
cyclin D1 protein. To determine whether the observed growth suppression
triggered by RA or a receptor-selective or receptor-nonselective
agonist was due to the induction of apoptosis, we performed terminal
deoxynucleotidyltransferase assays either after RA treatment or
independently after treatment with the RXR agonist SR11246. Appreciable
apoptosis was not detected at growth-suppressive concentrations of
either retinoid examined (data not shown).
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To explore whether the mechanism of the RARß- or RXR-mediated decrease in cyclin D1 involved cyclin D1 proteolysis, which is known to be activated by RA treatment of immortalized human bronchial epithelial cells (8), we treated BEAS-2B cells with 100 µM calpain inhibitor I, the inhibitor of the 26S proteasome pathway, with or without the addition of 1 µM RARß agonist (BMS-189453) or 2 µM RXR agonist (SR11246). A dose-dependent decline in cyclin D1 was observed after treatment with these agonists (Fig. 4
) and after treatment with the RXR agonist
SR11345 (data not shown). However, when cells were treated with calpain
inhibitor I and with the RARß- or RXR-selective agonist, calpain
inhibitor I inhibited the decline in the expression of cyclin D1
protein induced by the receptor-selective agonists (Fig. 4). Thus,
these retinoids mediate the decline of cyclin D1 through a proteolytic
pathway similar to that reported for RA (8). |
DISCUSSION |
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RA inhibits bronchial epithelial cell transformation in vitro(7), and clinical trials report that retinoids reduce some second primary cancers, including aerodigestive tract cancers (1,2). However, it is not clear why retinoids have cancer chemoprevention activity (3), whereas the examined carotenoids do not (4-6). RA signaling involves the RARs and RXRs. The RARs and RXRs are ligand-dependent transcription factors that have the potential to homodimerize and heterodimerize and are known to alter the expression of "downstream" species that directly mediate the biologic effects of retinoids. The regulation of cell cycle progression is also under active study and involves cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors [reviewed in (20-25)]. This report confirms and extends prior work that demonstrates that these two growth-regulatory programs, the cell cycle machinery and retinoid signaling, are coupled through post-translational regulation of cyclin D1, the G1-phase cyclin (7,8). Notably, treatment with retinoid receptor-nonselective agonists, but not
-carotene and
ß-carotene, led to growth suppression and a decrease in the
expression of cyclin D1 protein. The findings obtained with these
carotenoids may also relate to their cellular permeability properties
or to the ability of cells to generate active metabolites in culture.
These in vitro findings, however, parallel observations of the
clinical inactivity of these carotenoids in clinical cancer
chemoprevention trials (4-6). 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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Supported in part by Public Health Service grants R01CA54494-06 (E. Dmitrovsky), T32CA09512 and K12CA01712 (J. Langenfeld), and T32CA09685 (J. O. Boyle), National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by American Cancer Society grant RPG-90-019-08-DDC (E. Dmitrovsky); by the Byrne Fund (E. Dmitrovsky); by the Oracle Chemoprevention Research Fund (E. Dmitrovsky); and by the American Society of Clinical Oncology Young Investigator Award (J. O. Boyle).
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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