[CANCER RESEARCH 59, 4510 – 4515, September 15, 1999]
Advances in Brief
The Tobacco-specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Is
a b-Adrenergic Agonist and Stimulates DNA Synthesis in Lung Adenocarcinoma
via b-Adrenergic Receptor-mediated Release of Arachidonic Acid1
Hildegard M. Schuller,2 Patricia K. Tithof, Michelle Williams, and Howard Plummer III
Experimental Oncology Laboratory [H. M. S., M. W., H. P.] and Department of Animal Science [P. K. T.], College of Veterinary Medicine, University of Tennessee, Knoxville,
Tennessee 37996
Abstract
Lung cancer is the leading cause of death in the United States, and it
demonstrates a strong etiological association with smoking. The nicotinederived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) reproducibly induces pulmonary adenocarcinomas (ACs) in laboratory rodents and is considered an important contributing factor to the
high lung cancer burden observed in smokers. It has been shown that the
development of NNK-induced ACs in mice is reduced by inhibitors of
cyclooxygenase and lipoxygenase and that the growth of human AC cell
lines is regulated by b-adrenergic receptors. On the basis of structural
similarities of NNK with classic b-adrenergic agonists, we tested the
hypothesis that NNK stimulates the growth of human AC cells via
agonist-binding to b-adrenergic receptors, resulting in the release of
arachidonic acid (AA). In support of this hypothesis, radioreceptor assays
with transfected CHO cell lines stably expressing the human b1- or
b2-adrenergic receptor demonstrated high affinity binding of NNK
to each of these receptors. Two human AC cell lines expressed b1- and
b2-adrenergic receptors by reverse transcription-PCR and responded to
NNK with the release of AA and an increase in DNA synthesis. b-Adrenergic antagonists completely blocked the release of AA and increase in
DNA synthesis. The cyclooxygenase inhibitor aspirin and the 5-lipoxygenase inhibitor MK-886 both partially inhibited DNA synthesis in response
to NNK. Our findings identify the direct interaction of NNK with b-adrenergic, AA-dependent pathways as a novel mechanism of action which
may significantly contribute to the high cancer-causing potential of this
nitrosamine. Moreover, NNK may also contribute to the development of
smoking-related nonneoplastic disease via this mechanism.
Introduction
Lung cancer is the leading cause of cancer death in the United
States, with a mortality rate of .95% within 1 year of diagnosis (1, 2).
AC3 is the most common type of lung cancer (;60%; Ref. 3), and it
is highly resistant to current therapeutic strategies.
Smoking is the single most extensively documented risk factor for
all histological types of lung cancer, including AC (4). Among the
numerous toxic and carcinogenic agents contained in tobacco products, the nicotine-derived NNK is the most potent carcinogen in
laboratory animals and has, therefore, been implicated as a major
cause of tobacco-associated lung cancer (5). NNK reproducibly induces a high incidence of AC in laboratory rodents (6). In analogy to
Received 5/7/99; accepted 8/4/99.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by USPHS Grant RO1CA51211 from the National Cancer Institute (to
H. M. S.).
2
To whom requests for reprints should be addressed, at Experimental Oncology
Laboratory, College of Veterinary Medicine, University of Tennessee, 2407 River Drive,
Knoxville, TN 37996.
3
The abbreviations used are: AC, adenocarcinoma; NNK, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; cox, cyclooxygenase; AA, arachidonic acid;
125
[ I]CYP, [125I]iodocyanopindolol; RT, reverse transcription; M-MLV, Moloney murine
leukemia virus; NNN, N9-nitrosonornicotine.
the human disease (7, 8), these experimentally induced ACs express
activating point mutations in the Ki-ras gene that are thought to be
caused by the formation of promutagenic DNA adducts from reactive
NNK metabolites (9 –12).
We have shown that cell lines derived from human pulmonary AC
express b-adrenergic receptors and that classic agonists for these
receptors stimulate DNA synthesis in these cells (13). Moreover,
recent studies in a mouse model have demonstrated that inhibitors of
5-lipoxygenase and cox-1 and cox-2 reduced the incidence and multiplicity of NNK-induced ACs (14 –16), whereas studies in human
lung tumors have revealed high levels of cox-2 protein in welldifferentiated ACs (17). Although the predominant signal transduction
pathway activated by b-adrenergic receptors in most mammalian cells
involves the activation of adenylate cyclase and cyclic 39,59-AMP (18,
19), it has been shown that, in some cell systems, binding of agonist
to this receptor family can result in the release of AA (20 –22). AA
itself is an important second messenger that plays a role in the
regulation of cellular proliferation in smooth muscle cells through the
activation of several signaling pathways, which include mitogenactivated protein kinases and protein kinase C (23–25). Alternatively,
inhibition of metabolism of AA may increase AA concentrations, and
this may be important in the inhibition of cell proliferation. In a
previous study, nonsteroidal anti-inflammatory agents were shown to
inhibit colon tumorigenesis by increasing the levels of free AA,
which, in turn, stimulated the production of ceramides, a known
mediator of apoptosis (26). Moreover, AA serves as a precursor for
the generation of biologically active eicosanoids, including prostaglandins, thromboxanes, and leukotrienes (27, 28). The formation of
prostaglandins and thromboxanes involves metabolism of AA by
members of the cox family, whereas the generation of leukotrienes is
the result of AA metabolism by 5-lipoxygenases (28).
Because of structural similarities of NNK with classic b-adrenergic
agonists (Ref. 19; Fig. 1), we hypothesized that this carcinogenic
nitrosamine may be an agonist for this receptor family and may
activate an AA-dependent mitogenic signal transduction pathway via
ligand binding to these receptors in AC cells. In support of this
hypothesis, NNK bound with high affinity to stably expressed human
b1- or b2-adrenergic receptors in two transfected CHO cell lines and
caused the release of AA via ligand binding to these receptors in AC
cells, resulting in cox- and lipoxygenase-dependent stimulation of
DNA synthesis.
Materials and Methods
Cell Culture. The human AC cell lines NCI-H322 and NCI-H441 were
purchased from the American Type Culture Collection (Manassas, VA) and
maintained in RPMI supplemented with fetal bovine serum (10%, v/v), Lglutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 mg/ml) at
37°C in an atmosphere of 5% CO2. A CHO cell line (Rex 50) transfected with
the human b1 adrenoreceptor gene and stably expressing human b1 receptors
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NNK STIMULATES LUNG AC GROWTH VIA b-ADRENORECEPTORS AND AA
Fig. 1. Nicotine is a tertiary amine consisting of a pyridine and a pyrrolidine ring. NNN
is formed by nitrosation of the pyrrolydine ring, whereas the formation of NNK involves
nitrosation under ring opening of the pyrrolidine ring (11, 12, 48). The catecholamines
epinephrine and norepinephrine are comprised of a catechol ring with an aliphatic side
chain containing a nitrogen atom, and they are agonists for all a- and b-adrenergic
receptors (18). The intact catechol ring is a requirement for a- but not b-adrenergic
agonists. Increasing the steric bulk of the N-substituents, as in isoproterenol, increases the
selectivity for b-adrenergic receptors (18). The heterocyclic pyridine ring of NNK
resembles the catechol ring stereologically. Similar to classic b-adrenergic agonists such
as isoproterenol, NNK contains a nitrogen atom in an aliphatic side chain, the bulk of
which in isoproterenol is increased by two methyl groups or in NNK by the N-nitroso
group and a methyl group.
and a CHO cell line transfected with the human b2 gene and stably expressing
human b2-adrenergic receptors were kindly provided to us by Dr. R. J.
Lefkowitz (Duke University Medical Center, Durham, NC). These cell lines
were maintained in Ham’s F-12 medium supplemented with fetal bovine serum
(10%, v/v), L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin
(100 mg/ml) at 37°C in an atmosphere of 5% CO2.
Radioreceptor Assay. CHO cells or AC cells at 75% confluency were
washed twice with PBS, scraped off the culture vessels, resuspended in PBS,
and collected in ice-cold lysis buffer [5 mM Tris-5 mM EDTA (pH 7.4)]
containing the protease inhibitors benzamidine, soybean trypsin inhibitor, and
leupeptin (Sigma Chemical Co., St. Louis, MO), all at 10 mg/ml. Crude
membrane preparations were generated by centrifugation of the cell suspension
(10 min at 4°C at 19,000 rpm), followed by removal of the supernatant and
recentrifugation (12,000 rpm for 10 s) after the addition of fresh lysis buffer
(29). The resulting membranes were resuspended in 75 mM Tris, 5 mM MgCl2,
and 2 mM EDTA (pH 7.4) buffer containing protease inhibitors. Radioreceptor
assays (30) were conducted in which ascending concentrations of nonradioactive ligands (NNK, epinephrine, norepinephrine, atenolol, and ICI118,551)
competed with the b-radioligand [125I]CYP (at the concentrations indicated in
the figure legends; 2200 Ci/mmol; NEN, Boston, MA), under steady-state
conditions (incubation for 45 min at room temperature), established by saturation binding assays with the radioligand at various temperatures and for
various incubation times. Each assay consisted of 25 ml of competing ligand or
water, 25 ml of [125I]CYP, and 200 ml of crude membrane preparation (25 mg
of protein per assay tube; Ref. 31) and reaction buffer containing protease
inhibitors. Ascorbic acid (1 mM) was added to prevent oxidative breakdown of
the catecholamines (32), 5-hydroxytryptamine (10 mM) was added to prevent
binding of the radioligand to serotonin receptors (33), pargyline (10 mM) was
added to inhibit the metabolic conversion of the catecholamines by monoamine
oxidase (29), and 1-amino-benzotriazole (10 mM) was added to inhibit the
metabolic conversion of NNK by cytochrome P450 (5, 11). Nonspecific
binding was determined by incubations in the presence of alprenolol (1 mM;
RBI, Natick, MA). The reaction was terminated by the addition of 2 ml of
ice-cold Tris buffer (10 mM) and collection of bound radioactivity on Whatman
GF/C filters by vacuum filtration (Brandel cell harvester). Following three
washes with Tris buffer in the harvester, radioactivity bound to the filters was
determined with a gamma counter (Packard, Meriden, CT).The binding data
were analyzed by nonlinear regression for single-site or two-site isotherms
using a computer program (Ref. 34; Prism/GraphPad for the Macintosh).
RT-PCR. RNA was isolated from NCI-H322 or NCI-H441 cells using
guanidine isothiocyanate-cesium chloride ultracentrifugation (35). Concentration of the RNA was determined by absorbance at 260 nm.
For the RT reaction, 2 mg of RQ1-treated RNA and 1 mg of oligo(dT)12–18
primers (Life Technologies, Inc., Grand Island, NY) in nuclease-free water
were heated to 82°C for 3 min and then placed on ice. To this solution was
added 0.5 mM dNTPs, 10 mM DTT, 40 units of RNasin RNase inhibitor
(Promega, Madison, WI), 200 units of M-MLV reverse transcriptase (Life
Technologies, Inc.), and 103 buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl,
and 15 mM MgCl2], in a final volume of 20 ml. The reaction mixture was
incubated at 37°C for 1 h, followed by heat inactivation for 10 min at 92°C. A
negative control reaction was performed without the M-MLV.
The PCR was performed with 5 ml of the RT reaction, which was mixed
with 0.2 mM dNTPs, 5 ml of 103 PCR buffer [100 mM Tris-HCl (pH 8.3), 500
mM KCl, and 15 mM MgCl2], 1.25 units of SuperTaq polymerase (Ambion,
Austin, TX), 5% DMSO, a primer pair for cyclophilin used as an internal
control (75 or 125 nM; Ambion), primers for the human b1 or b2-adrenergic
receptors (250 or 500 nM) and nuclease-free water in a final volume of 50 ml.
The b1-adrenergic receptor primers (forward, 59-caagtgctgcgacttcgtcacc-39;
and reverse, 59-gccgaggaaacggcgctc-39) amplified a 159-bp fragment (36). The
PCR conditions for the b1 primers were: 1 cycle of 2 min at 94°C; 35 cycles
of 94°C for 45 s, 55°C for 45 s, and 74°C for 45 s; and a final extension for
5 min at 74°C. The b2-adrenergic receptor primers (forward, 59-acgcagcaaagggacgag-39; and reverse, 59-cacaccatcagaatgatcac-39) amplified a 401-bp fragment (37). The PCR conditions for the b2 primers were: 1 cycle of 2 min at
94°C; 37 cycles of 94°C for 60 s, 56°C for 60 s, and 72°C for 60 s; and a final
extension for 5 min at 72°C. Reactions were run on a MJ Research (Watertown, MA) PTC-200 thermal cycler.
One-half of the PCR (25 ml) was run on a 1.5% agarose (Life Technologies,
Inc.) gel for 2.15 h at 75 V. A 100-bp DNA ladder (Life Technologies, Inc.)
was run on the same gel. The gel was imaged by ethidium bromide staining
using a UVP (Upland, CA) GDS 7500 or an Ultra Lum (Paramount, CA)
TUI-5000 gel documentation system.
The PCR fragments were sequenced using the forward primers used to
amplify the fragment by RT-PCR with the ABI Terminator Cycle Sequencing
reaction kit on an ABI 373 DNA sequencer (Perkin-Elmer, Foster City, CA).
Sequences were entered into DNASIS software (Hitachi, South San Francisco,
CA). The sequences using the forward primers were compared with the
sequence of human b1-adrenergic receptor (GenBank accession no. J03019,
bases 747– 887) or b2-adrenergic receptor (GenBank accession no. M15169,
bases 1677–2060).
Determination of AA Release from Prelabeled AC Cells. The release of
AA by AC cells was determined as described previously (38). Briefly, NCIH322 or NCI-H441 cells were seeded into six-well plates (105 cells/well) in
complete RPMI. When they had reached 75% confluency (3 days later), the
cells were incubated with [3H]AA (0.25 mCi/ml; specific activity, 200 –240
Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) for 24 h. Following two washes with medium containing 0.1% BSA, cells were incubated
in medium with 0.1% BSA for 45 min. BSA was added to the medium to trap
released fatty acids, thus inhibiting subsequent metabolism and reacylation.
Accordingly, the radioactivity in the supernatant reflected cumulative deacylation of [3H]AA from phospholipid pools. Preliminary experiments on AA
release over time in response to NNK (1 mM) established that maximum release
occurred after 2 min of incubation (data not shown). This time interval was
then chosen to study concentration dependence of this effect as well as
modulation of the AA response by preincubation (10 min) with the b1adrenergic antagonist atenolol or the b2-adrenergic antagonist ICI118,551.
Following incubation, the medium was removed and placed into scintillation
vials with scintillation cocktail (Microscint, 20 ml). The remaining monolayers
were dissociated by 103 trypsin-EDTA (1 ml) and placed into scintillation
vials with scintillation cocktail (20 ml). Radioactivity was determined by liquid
scintillation spectrophotometry (Top Count; Packard). Released AA was expressed as percent of total incorporated cellular AA. Statistical analysis of data
was performed by ANOVA, and group means were compared using the
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NNK STIMULATES LUNG AC GROWTH VIA b-ADRENORECEPTORS AND AA
Fig. 2. Results of radioreceptor assays with cell membrane fractions from CHO cell
line Rex 50 transfected with the human b1-adrenenergic receptor gene and stably expressing human b1-adrenergic receptors. Ascending concentrations of NNK, the physiological agonist with preference for b1 receptors, norepinephrine, or the site-selective b1
antagonist, atenolol, competed for b1 binding sites with [125I]CYP (100 pM). The affinity
of NNK to this receptor was 2.5 times greater than that of atenolol and 602 times greater
than the affinity of norepinephrine (EC50s, calculated by nonlinear regression for single
site-binding isotherms, were: NNK, 5.8 nM; atenolol, 14.0 nM; and norepinephrine, 3.49
mM).
Student-Newman-Keul test. Appropriate transformations were performed on
all data that did not follow a normal distribution.
[3H]Thymidine Incorporation Assays. Cells were seeded into 96-well
plates (5 3 103 cells/well, triplicate wells per treatment group) in complete
RPMI and allowed to settle for 4 h. [3H]Thymidine (76 Ci/mmol, 0.5 Ci/well;
Amersham) and NNK (at the concentrations listed in the figure legends) were
then added. The b-adrenergic antagonist propranolol, the cox-inhibitor aspirin,
or the 5-lipoxygenase-inhibitor MK-886 was added immediately prior to NNK
at the concentrations specified in the figure legends. Following an incubation
period of 24 or 48 h (as specified in the figure legends) in an atmosphere of 5%
CO2 at 37°, the cells were washed twice with PBS, followed by 0.1 N NaOH.
Scintillation fluid (Microscint) was then added to the wells, and radioactivity
was measured with a microplate scintillation and luminescence counter (Top
Count). Assays under identical conditions but using numbers of viable cells by
hemocytometer after trypan blue dye exclusion stain as end point were conducted in parallel. Statistical evaluation of data were by nonparametric
ANOVA, Mann-Whitney U test, or unpaired Student’s t test.
Results and Discussion
Radioreceptor assays with CHO cell line Rex 50, which stably
expresses the human b1-adrenoreceptor, showed that NNK competed
with high affinity for b1-binding sites against [125I]CYP (Fig. 2).
Under the assay conditions used here, the affinity of NNK
(EC50 5 5.8 nM) to this receptor was higher than that of the siteselective b1-antagonist atenolol (EC50 5 14.0 nM) or the physiological agonist norepinephrine (EC50 3.49 mM). Similarly, NNK competed with high affinity for b2-adrenergic binding sites against
[125]CYP in CHO cell line NBR29 transfected with the human b2
adrenoreceptor gene (Fig. 3). The affinity of NNK (EC50 5 128 nM)
to this receptor was higher than that of the site-selective b2 antagonist
ICI118,551 (EC50 5 2.91 mM) or the physiological agonist epinephrine (EC50 5 278 mM). Because the affinity of NNK to the b1 receptor
was about 22 times higher than its affinity to the b2 receptor, it is
expected that, in the presence of both receptor types, NNK will
preferentially bind to the b1 receptor. NNK also competed with high
affinity with [125I]CYP for b-adrenergic binding sites in the AC cells
but yielded a shallower curve, suggesting the presence of more than
one binding site (see Fig. 5, inset). Accordingly, atenolol and
ICI118,551, each of which is highly site-selective for only one type of
b-adrenergic receptor (atenolol for b1, ICI118,551 for b2 receptors)
and binds to other b receptors only at relatively high concentrations,
yielded a clearly biphasic binding curve in AC cells (see Fig. 5, inset).
Analysis of the binding data by nonlinear regression for a two-site
binding isotherm identified 60% of the receptors present as b1 and
40% as b2 receptors. Predictably, the nitrosamine NNN did not bind
to b1- or b2-adrenergic receptors. This finding is in accord with
structural characteristics of NNN, which is formed from nicotine by
nitrosation of the pyrrolidine ring (Fig. 1) and lacks the aliphatic side
chain containing a nitrogen atom formed by ring-opening of the
pyrrolidine ring and nitrosation during the formation of NNK from
nicotine (Fig. 1).
In accordance with our receptor binding data, the human AC cell
lines NCI-H322 and NCI-H441 both expressed mRNA for b1 and
b2-adrenergic receptors by RT-PCR, with b1 mRNA yielding the
more prominent band (Fig. 4). The PCR fragments amplified by the
human b1 primers in the AC cells were 100% identical to the published sequence (GenBank accession no. J03019, bases 747– 887).
The PCR fragments amplified by the human b2 primers in AC cells
were 100% identical to the published sequence (GenBank accession
no. M15169, bases 1677–2060). These findings are in accordance
Fig. 3. Results of radioreceptor assays with cell membrane fractions of CHO cell line
NBR29 transfected with the human b2-adrenergic receptor gene and stably expressing
human b2-adrenergic receptors. Ascending concentrations of NNK, the physiological
agonist with preference for b2 receptors, epinephrine, or the site-selective b2 antagonist,
ICI118,551, competed for b2 binding sites with [125I]CYP (100 pM). The affinity of NNK
to this receptor was 22.7 times greater than that of ICI118,551 and 2172 times greater than
the affinity of epinephrine (EC50s, calculated by nonlinear regression for single sitebinding isotherms, were: NNK, 128 nM; ICI118,551, 2.91 mM; and epinephrine, 278 mM).
Fig. 4. Expression of mRNA for b1- and b2-adrenergic receptors in the human AC cell
lines NCI-H322 and NCI-H441 by RT-PCR. The b1 primers amplified a 159-bp fragment,
whereas the b2 primers amplified a 401-bp fragment. Lane 1, NCI-H322 with b2 primers;
Lane 2, NCI-H441 with b2 primers; Lane 3, NCI-H441 with b2 and cylophylin primer;
Lane 4, NCI-H441 with cylophylin primer alone; Lane 5, NCI-H322 negative control
without M-MLV reverse transcriptase; Lane 6, NCI-H322 with b1 primers; Lane 7,
NCI-H441 with b1 primers; Lane 8, NCI-H322 with b1 and cylophylin primer; Lane 9,
NCI-H322 negative control without M-MLV reverse transcriptase; Lane M, a 100-bp
DNA ladder.
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NNK STIMULATES LUNG AC GROWTH VIA b-ADRENORECEPTORS AND AA
Fig. 5. Results of AA release assay in NCI-H322
cells. NNK caused a concentration-dependent release
of AA with a maximum release observed with 10 nM
NNK. The response to 10 nM NNK was significantly
(P , 0.005) inhibited by atenolol (1 mM) and
ICI118,551 (1 mM), indicating that the release of AA
was mediated by ligand binding of NNK to b1- and
b2-adrenergic receptors. Similar results were obtained
with NCI-H441 cells. Inset, results of radioreceptor
assays in AC cell line NCI-H322. Because one objective of these assays was to assess the relative proportions of b1- and b2-adrenergic receptors in these
cells, these assays were conducted with the saturation
concentration of [125I]CYP (300 pM) to ensure complete b receptor occupancy with radioligand. In accordance with our findings in the transfected CHO
cell lines (Figs. 2 and 3), which identified NNK as a
high-affinity ligand for both b1- and b2-adrenergic
receptors, NNK yielded a shallow binding curve well
to the left of the curves generated by atenolol or
ICI118,551, suggestive of more than one high-affinity
binding site for NNK. By contrast, atenolol and
ICI118,551, each of which is highly site selective for
only one of these receptors (atenolol, b1; ICI118,551,
b2) generated clearly biphasic curves. Analysis of the
binding data by nonlinear regression for two-site
binding isotherms revealed that the b1-selective antagonist atenolol bound with high affinity to
60 6 3.0% and with low affinity to the remaining
receptors occupied by the radioligand, whereas the
b2-selective antagonist ICI118,551 bound with high
affinity to 40 6 4% and with low affinity to the
remaining receptors occupied by [125I]CYP.
with published data generated in radioreceptor assays, which had
revealed the presence of b-adrenergic receptors in these cell lines
(13).
NNK caused a concentration-dependent release of AA in both AC
cell lines (Fig. 5). AA release in response to 10 nM NNK was
significantly inhibited by preincubation with the b1 antagonist atenolol (1 mM; P , 0.005) or the b2 antagonist ICI118,551 (1 mM,
P , 0.005). These relatively high antagonist concentrations were used
to counteract the exceptionally high affinity of NNK to b1 and b2
receptors. As the selectivity of the antagonists may have been negatively affected in these assays, further studies with lower NNK concentrations are required to establish the role of each receptor type in
dose-response curves for each antagonist. Collectively, these findings
identify NNK as a high-affinity agonist for b1- and b2-adrenergic
receptors, with activation of the AA cascade as the downstream
effector in human pulmonary AC cells. Further studies are clearly
warranted to delineate more clearly the role of each b-adrenergic
receptor, to identify which products are formed from AA in these
cells, and to determine how these products may affect intracellular
signaling events.
Analysis of DNA synthesis in AC cells by the incorporation of
[3H]thymidine as well as cell counts by hemocytometer demonstrated
a significant (P , 0.001) stimulation by NNK (10 nM; Fig. 6). This
effect was completely inhibited by the broad-spectrum b-adrenergic
antagonist propranolol (1 mM, P , 0.001) and partially inhibited by
the cox-inhibitor aspirin (100 mM; P , 0.05) and the lipoxygenaseinhibitor MK-886 (10 mM; P , 0.001 Fig. 6). These data suggest that
binding of agonist NNK to b1- and b2-adrenegic receptors in AC cells
Fig. 6. Results of [3H]thymidine incorporation
assay in NCI-H322 cells. The NNK concentration
(10 nM), which had yielded maximum release of
AA (Fig. 5), significantly stimulated DNA synthesis. This effect was partially inhibited by the coxinhibitor aspirin and the lipoxygenase-inhibitor
MK-886 and was completely blocked by the broadspectrum antagonist of b-adrenergic receptors, propranolol. Inset, analysis of viable cell numbers after
trypan blue dye exclusion stain by hemocytometer
following a 48-h incubation period of NCI-H441
cells with a low concentration (1 nM) of NNK and
the effect of atenolol or ICI118,551 (1–1000 nM) on
NNK-induced increase in cell number. The b1 antagonist atenolol more potently inhibited the response to NNK, possibly reflecting the relative
predominance of b1-adrenergic receptors in AC
cells. Columns, means of triplicate samples per
treatment group; bars, SD.
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NNK STIMULATES LUNG AC GROWTH VIA b-ADRENORECEPTORS AND AA
activated an AA-dependent mitogenic signal transduction cascade that
involved both cox- and lipoxygenase-dependent messengers. The
observed NNK-induced increase in cell numbers were consistently
greater than the increase in DNA synthesis, suggesting that mechanisms in addition to stimulation of DNA synthesis (e.g., inhibition of
apoptosis) may be involved. Assays using a lower concentration of
NNK (1 nM) in the presence of atenolol or ICI118,551 ranging in
concentration from 1 nM to 1 mM identified atenolol as the more potent
inhibitor of cell proliferation (Fig. 6, inset). Future studies will need
to clarify whether the observed difference in antagonist efficacy is a
reflection of the relative predominance of b1-adrenergic receptors in
AC cells or whether they are caused by the generation of different AA
products.
Our findings implicate binding of NNK as an agonist to b1- and
b2-adrenergic receptors and the resulting activation of the AA cascade
as cellular events contributing to the development of pulmonary AC in
smokers. This interpretation is in accordance with recent reports on
the activation of a ras and Src tyrosine kinase-dependent mitogenactivated protein kinase pathway by b2-adrenergic receptors in fibroblasts (39). Because reactive NNK metabolites cause activating point
mutations in the ras gene (9, 10), an additional activation of ras by
binding of NNK as an agonist to b-adrenergic receptors likely potentiates the mitogenic response. Further studies are clearly needed to
dissect the complex signaling events involved.
Our data suggest that b-adrenergic antagonists may prevent the
continuous growth stimulation that contributes to the development of
this histological lung cancer type in smokers chronically exposed to
NNK, whereas inhibitors of cox or lipoxygenase may be only partially
effective. However, these enzyme inhibitors may have additional
cancer-preventive effects by reducing the metabolic activation of
NNK. This interpretation is supported by recent reports that have
shown the metabolic activation of NNK by members of the cox or
lipoxygenase families (15, 16, 4, 40). Broad-spectrum b-adrenergic
antagonists such as propranolol or alprenolol as well as b1 antagonists
such as atenolol are widely used for the therapy of hypertension (41)
and atherosclerosis (42, 43). Moreover, the broad-spectrum cox-inhibitor aspirin as well as b-blockers are widely used for the therapy
and prevention of heart attacks (43, 44). Because these cardiovascular
diseases are among the many adverse health effects caused by smoking (45, 46), epidemiological data to determine the chemopreventive
effects of these agents on AC development should be readily available.
Apart from the obvious significance of our findings for the genesis
and potential cancer intervention of pulmonary AC, the fact that NNK
is a high-affinity agonist for b1-and b2-adrenergic receptors additionally implicates this tobacco-specific nitrosamine in the etiology of
smoking-related cardiovascular disease. This nonneoplastic disease
complex has traditionally been attributed primarily to an increased
release of catecholamines in response to nicotine (22, 46, 47). On the
other hand, it has been shown that smoking reduces the efficacy of
b-blockers as antihypertensive agents (46). In light of our data, a
plausible explanation for this phenomenon is that, in smokers, these
agents must compete with NNK for b-adrenergic binding sites. Further studies are clearly needed to address this important issue.
Acknowledgments
We gratefully acknowledge the invaluable help of Dr. R. J. Lefkowitz (Duke
University Medical Center), who provided the transfected CHO cell lines and
advised us on radioreceptor assays. We also thank Dr. D. A. Schwinn (Duke
University Medical Center) for her advice on the RT-PCR assays and Dr. N.
Quigley (University of Tennessee, Sequencing Laboratory) for his assistance
with the sequencing.
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The Tobacco-specific Carcinogen
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Is a β
-Adrenergic Agonist and Stimulates DNA Synthesis in Lung
Adenocarcinoma via β-Adrenergic Receptor-mediated Release
of Arachidonic Acid
Hildegard M. Schuller, Patricia K. Tithof, Michelle Williams, et al.
Cancer Res 1999;59:4510-4515.
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