Cytokine-induced nitric oxide formation in normal but not in
neoplastic murine lung epithelial cell lines
David C.
Thompson1,
Stephanie
E.
Porter1,
Alison K.
Bauer2,
Kumuda C.
Das3,
Brandon
Ou1,
Lori
Dwyer-Nield1,
Carl W.
White3, and
Alvin M.
Malkinson1
Departments of
1 Pharmaceutical Sciences and
2 Pharmacology, University of
Colorado Health Sciences Center, Denver 80262-0238; and
3 Department of Pediatrics,
National Jewish Center for Immunology and Respiratory Medicine,
Denver, Colorado 80206
 |
ABSTRACT |
Cytomix, a mixture of interferon-
, tumor
necrosis factor-
, and interleukin-1
, induces nitric oxide (NO)
production in lung epithelial cell lines. It is not known whether
neoplastic transformation alters a cell's ability to form NO in
response to cytokines. The present study investigated NO formation in
two murine lines of immortalized "normal" (nontumorigenic) lung
epithelial cells of alveolar type II origin, E10 and C10, and their
sibling spontaneous transformants, E9 and A5. Nontumorigenic cells
elaborated much more NO after cytomix exposure than did their
tumorigenic counterparts. NO production was prevented by inhibiting
protein synthesis and NO synthase and attenuated by dexamethasone.
Northern and Western blot analyses of inducible NO synthase (iNOS)
demonstrated cytomix-induced induction of iNOS only in nontumorigenic
cells. The deficiency in NO production in tumorigenic cells was not
associated with reduced iNOS mRNA stability or with differences in
cytomix-induced nuclear factor-
B activation. Although cytomix caused
a greater production of NO in E10 cells than in E9 cells, the same
treatment induced equivalent proliferation in both cell lines. These
results indicate a specific deficiency in cytokine-induced NO synthesis in transformed murine lung epithelial cells relative to their normal
progenitor cells and provide a model for investigating iNOS regulation.
neoplasia
 |
INTRODUCTION |
NITRIC OXIDE (NO) exerts important physiological and
pathophysiological actions ranging from vascular relaxation and
neurotransmission to mediating macrophage cytotoxicity (44). Indeed,
many of the toxic effects of macrophages directed against tumor cells
involve NO, with inhibition of both mitochondrial respiration and DNA replication as important mechanisms (34, 36, 57). Investigation of the
role of endogenous NO in pulmonary pathophysiology is of great interest
because concentrations of NO are elevated in the air expired by
asthmatics (30) and by patients suffering from upper respiratory tract
infections (31) or bronchiectasis (32). Immunohistochemical
localization of inducible NO synthase (iNOS) in the pulmonary
epithelium (25) indicates local generation as a potential source of
exhaled NO. NO can exert either beneficial or deleterious effects
depending on the amount of NO and its biological context. A great
variety of cell types, ranging from macrophages to vascular smooth
muscle cells, can be induced to produce NO in response to cytokine
exposure (44). A mixture of interferon-
, interleukin-1
, and tumor
necrosis factor-
(empirically termed cytomix) is commonly used to
promote NO generation. Cytomix evokes NO production in immortalized and
neoplastic lines of bronchial and pulmonary epithelial cells (3, 26,
52, 53) and in primary cultures of human bronchial epithelial cells (3,
52).
In airway and lung epithelial cells, interleukin-1
and tumor
necrosis factor-
induce nuclear translocation of nuclear factor-
B (NF-
B; see Refs. 12, 28, 51). This transcription factor appears to
be an important inducer of iNOS expression (62, 63, 67). The
identification of several NF-
B binding motifs on the iNOS promoter
(66) supports this proposal. In addition, iNOS expression may be
regulated by other transcription factors, including interferon
regulatory factors (IRFs; see Ref. 37).
The role of NO in neoplasia remains to be clearly identified. Suggested
actions of endogenously produced NO in tumors include reduced
metastasis due to vasodilation and decreased platelet aggregation (15),
changes in cell proliferation (4, 11), and tumor angiogenesis (29).
Elevated levels of nitrite and nitrate, which are stable metabolites of
NO, were detected in bronchoalveolar lavage fluids of lung cancer
patients (2). Whether the NO is derived from tumor cells or activated
inflammatory cells remains to be defined. No studies have compared the
NO-producing capacity of related tumorigenic and nontumorigenic lung
epithelial cells. The E10 and C10 epithelial cell lines derived from
mouse lung expressed characteristics of type II epithelial cells at early passage (39). E9 and A5 cells are spontaneous transformants of
E10 and C10 cells, respectively, that produce tumors when injected into
mice (55). Other features that distinguish the tumorigenic siblings
from their nontransformed progenitors include lack of contact
inhibition of cell growth (54), expression of mutant Kras (46), and
constitutive expression of p53 (unpublished data). These cell lines
have been extensively characterized in order to understand the
biochemistry of pulmonary neoplasia (39).
The present study investigates the ability of cytomix and individual
cytokines to promote NO production in these sibling pairs of lung
epithelial cell lines. Our results indicate that cytokine-induced NO
production is deficient in the transformants.
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METHODS |
Cell culture and cytokine application.
E10 and C10 ("normal" or nontumorigenic cells) and E9 and A5
cells (tumorigenic sibling cells of E10 and C10, respectively) are of
type II lung epithelial cell origin, as originally described
by Smith and
colleagues (54, 55). Type
II features included the presence of lamellar bodies and the
biosynthesis of surfactant. E10 and C10 are contact inhibited and
anchorage dependent, whereas E9 and A5 are not. Cells were grown in
culture in CMRL-1066 medium containing 10% fetal bovine serum and
penicillin G (100 U/ml)-streptomycin (100 µg/ml)-amphotericin B (0.25 µg/ml) in a humidified atmosphere of 5%
CO2 in air on either 100-mm
plastic plates containing 10 ml of medium or 96-well plates containing
200 µl of medium. After confluence was attained, cells were serum
deprived for 24 h before cytokine administration and throughout the
48-h course of the experiment to prevent exposure to any agents present
in the serum that could modify iNOS expression. We determined in
preliminary studies that removal of serum for at least 8 h was
necessary to permit production of NO. Interferon-
(murine, 15 U/ng;
Sigma Chemical), tumor necrosis factor-
(human, 10 U/ng; Sigma
Chemical), and interleukin-1
(murine, 10 U/ng; Genzyme) were applied
singly or in combination to the medium. Cytomix is the combination of
all three cytokines at a final concentration of 10 ng/ml each. After
cytokine application, 100-µl samples of medium were withdrawn at
various times and stored frozen for later nitrite assay.
Estimation of cellular NO production.
Increases in medium concentration of nitrite were used as a measure of
NO production. This was validated by demonstrating the sensitivity of
cytomix-induced nitrite production to inhibition by the NO synthase
inhibitors aminoguanidine and monomethyl-L-arginine (Sigma
Chemical). In other experiments, glucocorticoid regulation of NO
production in E10 cells was examined by application of dexamethasone
(American Reagent Laboratories). Each agent was administered
concurrently with cytomix to produce the desired medium concentration.
Nitrite concentrations were determined in 20- to 100-µl aliquots
using chemiluminescence detection of NO as described previously (27). Briefly, medium samples were introduced into a stream of helium (ultrapure)-degassed reducing solution (0.1 M sodium iodide in 0.1 M
phosphoric acid). NO produced in the aqueous phase was released through
gas-permeable tubing in the Dunham cell and swept by a stream of
ultrapure helium (General Air) into an NO chemiluminescence detector
(Sievers 270B). Peak areas were recorded on an integrator (Hewlett-Packard 3390A), and sample concentrations were determined by
interpolation from a sodium nitrite standard curve. Nitrite levels for
each treatment were pooled (4-8 replicates) and represented as
means with associated SE. Comparisons between control and
cytokine-treated cells were made by Student's unpaired
t-test, with
P < 0.05 being significant.
Western blot analysis. Cellular levels
of iNOS protein were estimated by immunoblotting. Twenty-four hours
after cytomix application, cells were harvested by scraping and
sonicated in an ice-cold homogenization buffer consisting of 20 mM
HEPES-10% glycerol buffer, pH 7.5, and a mixture of protease
inhibitors [2 mM EDTA, 2 mM EGTA, 10% glycerol, 5 µg/ml
aprotinin, 10 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride
(PMSF)]. The sonicate was centrifuged at 16,000 g for 30 min, and the supernatant
(cytosol) was collected and mixed with sample loading buffer (0.1 M
Tris, pH 6.8, 2% SDS, 30% glycerol, 2%
-mercaptoethanol, and 2.5 mg/ml pyronine Y). Protein concentrations were determined by the method
of Lowry et al. (38). Aliquots containing 50 µg protein were
subjected to 7.5% polyacrylamide gel electrophoresis and transferred
to an Immobilon-polyvinylidene difluoride membrane (Millipore). These membranes were blocked with 0.2% casein plus 0.02% sodium azide in 15 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 (TNS buffer, blocking
buffer) for 60 min and subsequently incubated with a specific iNOS
mouse monoclonal antibody (Transduction Laboratories) at a 1:2,000
dilution for 60 min at 25°C. Blots were rinsed five times for 5 min
with wash buffer (0.1% Tween 20 + 1% milk in TNS) and incubated with
horseradish peroxidase-conjugated goat anti-mouse antibody
(Transduction Laboratories) as a secondary antibody for 60 min at
25°C. After additional rinses with wash buffer, immunoblots were
visualized by application of enhanced chemiluminescence Western blotting reagents (Amersham) to the blot and exposure to
autoradiographic film. A single band was identified as iNOS by
comparison with a macrophage lysate standard (Transduction
Laboratories) and molecular weight markers.
Northern blot analysis. Cellular
concentrations of iNOS mRNA were estimated by Northern blotting. Murine
iNOS cDNA cloned into pUC19 was generously provided by R. A. Robbins
(University of Nebraska). Plasmids were amplified in
Escherichia coli and purified with a
plasmid preparation kit (QIAGEN, Chatsworth, CA). cDNAs were isolated
from the vectors by treatment with Nco
I, gel purified, and labeled with a random-prime DNA labeling kit (BRL). Total RNA was isolated from cells using an RNeasy kit (QIAGEN) and quantitated spectrophotometrically. Standard molecular biology protocols (7) were followed. Fifteen micrograms of RNA were subjected
to electrophoresis on a 1% agarose-2.5 formaldehyde gel using a buffer
containing 20 mM 3-(N-morpholino)propanesulfonic acid and 1 mM EDTA (pH 7.4). RNA was tranferred to a nylon membrane (MSI) in
10× SSC buffer (1.5 M NaCl and 0.15 M sodium citrate) and
prehybridized for 30 min at 65°C in buffer containing 5× SSC, 5× Denhardt's, 0.5% SDS, and 0.1 mg/ml salmon sperm DNA. Blots were hybridized overnight with iNOS cDNA labeled to a specific activity
of 108
counts · min
1 · µg
1
using
[
-32P]cytidine
5'-triphosphate in prehydridization buffer at
65°C. The blots were washed three
times in a solution containing 2× SSC and 0.1% SDS and finally
rinsed in a solution of 0.2× SSC and 0.1% SDS. All washes were
conducted at 65°C. Blots were processed on a phosphorimager
(Molecular Dynamics).
In these studies, cells were prepared for Northern blot analysis 24 h
after treatment with cytomix or medium as a control. To evaluate RNA
stability, cells exposed to cytomix for 24 h were treated with 100 µM
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB), a
transcriptional inhibitor, and prepared for Northern blot analysis 3, 6, 9, or 12 h thereafter.
NF-
B nuclear
translocation. Nuclear extracts were obtained from
cells exposed to cytomix or a medium control for 2 h. Nuclear extracts
were prepared according to methods described previously (56) with the
following modifications. Approximately 2 × 106 cells were rinsed in 10 ml of
phosphate-buffered saline (PBS) and subjected to centrifugation (1,500 g for 5 min). The pellet was
resuspended in 1 ml of PBS and centrifuged (16,000 g for 15 s). The supernatant was removed, and the pellet was resuspended gently
in 400 µl of 10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 2 mM
dithiothreitol (DTT), 1 mM PMSF, 0.5 mg/ml leupeptin, and 0.3 mg/ml
antipain. After a 15-min period during which cells were allowed to
swell on ice, 25 µl of 10% Nonidet P-40 were added, and the tube was
vortexed for 10 s. The lysate was centrifuged for 30 s in a
microcentrifuge, and the nuclear pellet was resuspended in 20 mM HEPES,
pH 7.8, 0.42 M NaCl, 5 mM EDTA, 1 mM PMSF, and 10% (vol/vol) glycerol.
The tube was then rocked gently on a shaking platform for 30 min at
4°C, and this nuclear extract was centrifuged in a microcentrifuge
for 10 min at 4°C. The supernatant was collected and stored at
70°C for later electrophoretic mobility shift assay (EMSA).
Protein was measured using the Bradford protein assay (Bio-Rad; see
Ref. 5).
An NF-
B-specific oligonucleotide containing two tandemly arranged
NF-
B binding sites
(5'-GATCCAAGGGGACTTTCCATGGATCCA
CCATG3-') was synthesized (Applied Biosciences) for use in EMSA.
Two complementary strands were annealed in 10 mM Tris (pH 7.5)
containing 1 mM EDTA and end labeled using T4 polynucleotide kinase
(GIBCO) and
[
-32P]ATP (ICN) in
10× kinase buffer [0.5 M Tris · HCl (pH
7.5), 0.1 M MgCl2, 50 mM DTT, 1 mM
spermidine, and 1 mM EDTA]. Labeled double-stranded oligonucleotide was separated from free
[
-32P]ATP using a
G-50 Sephadex spin column (5 Prime
3 Prime). The binding
reaction was conducted according to a modified method of Garner and
Revzin (20) without labeled oligonucleotide. The nuclear extract was
maintained at 4°C for 15 min and incubated with labeled
oligonucleotide for 20 min at room temperature. The binding reaction
contained 10 µg of sample protein and 5× incubation buffer
[20% glycerol, 5 mM MgCl2,
5 mM EDTA, 5 mM DTT, 500 mM NaCl, 50 mM Tris · HCl
(pH 7.5), and 0.4 mg/ml calf thymus DNA]. Nuclear
protein-32P oligonucleotide
complex was separated from free
32P-labeled oligonucleotide by
electrophoresis through a 6% native polyacrylamide gel using a running
buffer consisting of 25 mM Tris (pH 8.0), 22.5 mM borate, and 0.25 mM
EDTA. In competition studies, 3.5 pmol of unlabeled NF-
B-specific
oligonucleotides were included in the binding reaction mixture.
Cell growth. The effect of cytokines
on cell proliferation was estimated by
5-(3-carboxylmethoxyphenyl)-2-(4-5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS) assay (CellTiter 96; Promega). This is a
colorimetric assay that measures the cellular conversion of MTS to
formazan, which absorbs at 490 nm (22). Validity of a similar (MTT)
assay in the measurement of growth of lung epithelial cell lines has
been shown previously (9). Twenty-four hours after cytomix application,
the medium was exchanged with RPMI medium containing fetal bovine serum
(10%), antibiotics (see above), and MTS reagents, and the cells were
incubated for 90 min. Medium absorbance was then read at 490 nm in a
96-well plate reader (Thermomax; Molecular Devices).
 |
RESULTS |
Application of cytomix to the cell lines generated NO, with the amount
of NO being cell line dependent. The nontransformed cells E10 and C10
produced more NO (34- to 78-fold) than did their respective neoplastic
siblings E9 and A5 (1- to 13-fold; Fig. 1).
NO production was estimated by measurement of elevations in medium
concentration of nitrite. The validity of this procedure was verified
by demonstrating that increases in nitrite induced by cytomix
administration to cells were inhibited by the NO synthase antagonists
aminoguanidine and monomethylarginine (Fig.
2). We characterized this induction
further. Concurrent treatment of cells with the protein synthesis
inhibitor cycloheximide prevented production of NO (Fig.
3). We infer that this reflects a
requirement for iNOS synthesis. Concentrations of dexamethasone as low
as 1 µM attenuated both basal and cytomix-induced NO generation (Fig. 4).

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Fig. 1.
Cell line specificity of cytomix-induced increases in nitrite
production. Normal (E10, C10; filled bars) and transformed (E9, A5;
open bars) lung epithelial cell lines were exposed to cytomix or medium
(vehicle control) for 24 h. Nitrite concentrations in the medium of
cytomix-exposed cells are expressed as a quotient of concentrations in
vehicle-treated cells. Nitrite levels in the medium of vehicle-treated
cells represented basal or constitutive production and were E10,
1.51 ± 0.05; E9, 1.03 ± 0.03; C10, 0.54 ± 0.04;
and A5, 0.38 ± 0.1 µM (mean ± SE). Data represent means of
the degree of increase from 3 experiments. [nitrite],
Nitrite concentration.
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Fig. 2.
Inhibition of cytomix-induced nitric oxide (NO) production by NO
synthase antagonists. E10 cells were exposed to medium (control) or to
the combination of interferon- , tumor necrosis factor- , and
interleukin-1 (cytomix). Cytomix-treated cells were incubated in the
presence of varying concentrations of monomethylarginine or
aminoguanidine. Samples of medium were taken 48 h after cytokine
application and measured for nitrite. Data represent means with
associated SE from 4 experiments.
* P < 0.05, unpaired
Student's t-test, compared with
control.
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Fig. 3.
Inhibition of cytomix-induced NO production by protein synthesis
inhibition. E10 cells were exposed to medium (control) or to the
combination of interferon- , tumor necrosis factor- , and
interleukin-1 (cytomix) in the presence or absence of cycloheximide
(10 µM). Samples of medium were taken 48 h after cytokine application
and measured for nitrite. Data represent means with associated SE from
4 experiments. * P < 0.05, unpaired Student's t-test, compared
with control.
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Fig. 4.
Attenuation of cytomix-induced NO production by dexamethasone. E10
cells were exposed to medium (control) or to the combination of
interferon- , tumor necrosis factor- , and interleukin-1
(cytomix) in the presence or absence of 1 or 10 µM dexamethasone.
Samples of medium were taken 48 h after cytokine application and
measured for nitrite. Data represent means with associated SE from
3-4 experiments. * P < 0.05, unpaired Student's t-test,
compared with untreated control.
P < 0.05, unpaired
Student's t-test, compared with
medium-exposed, cytomix-treated cells.
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The production of NO induced by cytomix was time dependent, with
elevations occurring 8 h after cytomix application and continuing for
up to 48 h (Fig. 5). The amount of NO
elicited by the combination of cytokines (as cytomix) exceeded that
produced by any single cytokine alone. Interferon-
induced
significant NO production by E10, A5, and C10 cells (Figs. 5 and
6). In E10 cells, the amount of NO
generated by interferon-
was comparable to that elicited by cytomix
(Fig. 4). The effect of interferon-
in promoting NO formation was
concentration dependent in E10 cells, and significant increases were
observed at concentrations exceeding 0.1 ng/ml (Fig.
7). NO production was unaffected by tumor
necrosis factor-
or interleukin-1
except in E10 cells in which
tumor necrosis factor-
caused a modest induction of NO formation
(Figs. 5-7). Although transformed A5 cells produce about three
times as much NO as do E9 cells, this remains approximately 10-fold
less than their nontransformed, growth-regulated sibling counterpart
C10 (Fig. 6).

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Fig. 5.
Time course of NO production in E10 cells induced by cytokines. E10
cells were exposed to medium (control), interferon- (IFN; 10 ng/ml),
tumor necrosis factor- (TNF; 10 ng/ml), interleukin-1 (IL-1; 10 ng/ml), or their combination (cytomix). Samples of medium were taken
after various incubation times and measured for nitrite. Data represent
means with associated SE from 4 experiments.
* P < 0.05, unpaired
Student's t-test, compared with
nitrite levels in control (untreated) cells at same time point.
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Fig. 6.
Induction of NO production in E9
(A), A5
(B), and C10
(C) cells by cytokines. Cells were
exposed to medium (control), IFN (10 ng/ml), TNF (10 ng/ml), IL-1 (10 ng/ml), or their combination (cytomix). Samples of medium were taken 48 h after cytokine application and measured for nitrite. Note the
different y-axis scales. Data
represent means ± SE from 4 experiments.
* P < 0.05, unpaired
Student's t-test, compared with
nitrite levels in control (untreated) cells.
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Fig. 7.
Concentration dependence of cytokine-induced induction of NO production
by E10 cells. E10 cells were exposed to medium (open bar) or different
concentrations of IFN, TNF, or IL-1. Samples of medium were taken after
48 h incubation with the cytokines and measured for nitrite. Data
represent means ± SE from 4 experiments.
* P < 0.05, unpaired
Student's t-test, compared with
control (untreated) cells.
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To investigate the mechanism causing the decreased ability of the
transformants to synthesize NO, iNOS content was examined in cells
exposed to cytomix for 24 h. Under these conditions, iNOS expression
was observed in the cytosol of cytomix-exposed E10 and C10 cells. In
the tumorigenic cell lines, cytomix elicited a small induction of iNOS
in A5 cells, but none was observable in E9 cells (Fig.
8). These cell line differences in
the extent of iNOS protein induction correlated well with their
respective amounts of NO synthesis.

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Fig. 8.
Western blot analysis of inducible NO synthase (iNOS). E10, E9, C10,
and A5 cells were exposed to medium ( ) or cytomix (+) for 24 h.
Cytosolic fractions were subjected to Western blot analysis using mouse
monoclonal anti-iNOS antibodies. Labeled proteins had the same
retardation factor as the macrophage lysate (mac) standard, with a size
of ~130 kDa.
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Northern blot analysis revealed that 24 h of cytomix exposure caused a
much larger increase in iNOS mRNA in E10 cells than in E9 cells (Fig.
9). However, no differences were apparent
between these cell lines with respect to iNOS mRNA stability (Fig. 9). This was assessed by inhibiting RNA synthesis at a peak in cytomix induction of iNOS and analyzing the amount of iNOS mRNA at different times thereafter. Thus greater instability of the iNOS message does not
account for the decreased iNOS mRNA content in E9 cells. Further
mechanistic studies were conducted in E10 and E9 cells to address the
lack of iNOS mRNA in cytomix-treated E9 cells. NF-
B appears to be
involved in the actions of various cytokines (1, 12, 28, 51). Upon
activation, cytosolic NF-
B is released from binding to its
inhibitory component I
B and translocates into the
nucleus to transcribe genes (35). In both E10 and E9 cells, cytomix
induced the nuclear translocation of the transcription factor NF-
B
to an apparently equal extent (Fig. 10).
Thus a failure in NF-
B activation does not underlie the E9 cell
resistance to cytokine induction of NO.

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Fig. 9.
Cytokine induction of iNOS mRNA and its stability. E10 and E9 cells
were exposed to medium ( ) or cytomix (+) for 24 h. Cellular RNA
was subjected to Northern blot analysis using mouse iNOS cDNA.
Inset: relative levels of iNOS mRNA 24 h after treatment with medium ( ) or cytomix (+). Graph
demonstrates time course of iNOS mRNA after treatment of cells with 100 µM of the transcriptional inhibitor
5,6-dichloro-1- -D-ribofuranosylbenzimidazole applied 24 h after cytomix administration. In both cell lines, iNOS mRNA at each
time point is expressed as a percentage of that measured at time
(t) = 0.
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Fig. 10.
Cytomix-induced translocation of nuclear factor- B (NF- B) to the
nucleus. E10 and E9 cells were exposed to medium ( ) or cytomix
(+) for 2 h. Nuclear extracts were subjected to electrophoretic
mobility shift assays using radiolabeled NF- B-specific
oligonucleotides (see METHODS). Competition studies with
excess unlabeled NF- B-specific oligonucleotides resulted in the loss
of the NF- B bands (data not shown).
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To consider whether the lack of effect of cytomix on NO production in
the neoplastic cells was due to a general defect in cytokine
responsiveness or was specific for cytokine induction of iNOS, the
proliferative actions of cytomix on the cell lines was evaluated. In
these studies, cytomix caused a 40-50% increase in cell number in
both lines (Fig. 11). This proliferative
effect of cytomix was prevented by the iNOS inhibitor aminoguanidine and by dexamethasone (Fig. 12).

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Fig. 11.
Cytomix induces proliferation of E10 and E9 cells. E10 and E9 cells
were exposed to medium or cytomix for 24 h. The size of the cell
populations was evaluated spectrophotometrically using a
5-(3-carboxylmethoxyphenyl)-2-(4-5-dimethylthiazolyl)-3-(4-sulfophenyl)
tetrazolium (MTS) assay. In each experiment, the absorbance in
cytomix-treated cells is expressed as a percentage of the absorbance in
untreated (control) cells. Data were pooled and are presented as means ± SE from 3-9 experiments.
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Fig. 12.
Inhibition of cytomix-induced proliferation of E10 cells. E10 cells
were exposed to medium, cytomix, or cytomix + aminoguanidine (1 mM) for
24 h (top). In other experiments,
E10 cells were incubated with medium, cytomix, or cytomix + dexamethasone (1 µM) for 48 h
(bottom). Cell populations were
evaluated spectrophotometrically using an MTS assay. Data are presented
as means ± SE from 4 experiments.
* P < 0.05, unpaired
Student's t-test, compared with
control cells. P < 0.05, unpaired Student's t-test, compared
with cells exposed to cytomix alone.
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 |
DISCUSSION |
The function of NO generated by lung epithelial cells remains to be
defined. Insofar as NO exerts cytotoxic and cytostatic actions and is
induced by cytokines, a role in inflammation and/or cellular
defense against pathogens has been proposed (40, 53). A striking
feature of induced NO generation is the seeming cellular ubiquity with
which cytokines can promote this process. NO production may be educed
in most of the cell types along the pulmonary tree. Primary cultures
and immortalized lines of bronchial (3, 53) and pulmonary epithelial
cells (3, 26, 48, 52, 53) generate NO in response to the application of
cytomix with or without the bacterial endotoxin lipopolysaccharide. The
results of the present study are largely consistent with these previous investigations in that cytomix can induce NO formation in immortalized but nontumorigenic murine lung epithelial cell lines. However, sibling
cells displaying neoplastic characteristics were resistant to this
effect. The generation of NO was time dependent; the initial lag time
required between cytokine application and accumulation of significant
nitrite in the medium (~8 h) is consistent with the requirement for
synthesis of iNOS. The sensitivity of NO production to inhibition by
the protein synthesis inhibitor cycloheximide confirms the necessity
for iNOS synthesis in NO production.
The primary focus of the present study was to determine whether NO
generation was altered in transformed lung epithelial cells. Cytokine-induced NO production has been examined in cultured lung type
II epithelial cells derived by clonal selection (L2; see Refs. 16, 26),
by primary isolation from normal lung (48), and in type II cell lines
derived from lung tumors (A549, LA-4; see Refs. 3, 21, 52, 53).
Although all of these cell lines produce NO, it is difficult to compare
production in nontumorigenic (or normal) cells with that in tumorigenic
cells because the normal cells were isolated from a different species
(rat) than were the cells from tumors (A549, human; LA-4, mouse). The
cell lines used in the present study are derived from murine lung type
II epithelial cells. The advantage of these particular cells is that
the tumorigenic cells (E9 and A5) are spontaneous transformants of the
normal cells (E10 and C10, respectively) and are therefore close
genetic relatives. The neoplastic cells generated considerably less NO in response to cytomix than did their respective nontransformed progenitors. This indicates that the ability to produce NO was diminished after neoplastic transformation. Western blot analyses in
the cell lines confirmed the cytomix enhancement of iNOS expression in
nontumorigenic cells with little, if any, induction in the tumorigenic
cells. The deficiency of iNOS protein in tumorigenic cells after
cytomix treatment suggests that an insufficiency of NO synthase
cofactors such as tetrahydrobiopterin is unlikely to be a factor
underlying the difference in NO production by these cells. Northern
blot analyses in E10 (nontumorigenic) and E9 (tumorigenic) cells were
consistent with the Western blot data in that cytomix-induced iNOS mRNA
was reduced in E9 cells relative to E10 cells. This may be a
consequence of reduced iNOS gene transcription in E9 cells or
diminished iNOS mRNA stability. With respect to the latter, previous
investigations have demonstrated iNOS mRNA stability as a possible site
of modulation of NO synthesis (33, 47). To examine this possibility,
iNOS mRNA levels were examined in cells after inhibition of
transcription using DRB. These studies revealed that, despite the
different intracellular mRNA concentrations, iNOS mRNA stability was
comparable in E10 and E9 cells. Hence, the possibility that message
destabilization was responsible for diminished NO production by the
tumorigenic cells can be excluded.
NF-
B is an important transcriptional inducer of iNOS expression in a
variety of cells (62, 63, 67). Indeed, two NF-
B binding motifs are
present in the murine iNOS promoter (66). The two cytokines used in the
present study, interleukin-1
and tumor necrosis factor-
, have
been shown to induce NF-
B translocation into the nucleus (12, 28,
51). In the present study, cytomix induced equivalent nuclear
translocation of NF-
B in both E10 and E9 cells. As such, the
likelihood that a defect in translocation of NF-
B was responsible
for the diminished NO production by E9 cells seems remote. Putative
transcription factor consensus sequences identified in the iNOS
promoter region in murine macrophages include nuclear factor
interleukin-6 binding sites, an NF-
B site, and a tumor necrosis
factor response element (37). Potential
trans-activating factor binding sites
have been located in a distal enhancer region and include an NF-
B
site and several IRF binding elements (37). Interleukin-6 had no effect
on NO production in E10 cells (unpublished observation).
Interferon-
, on the other hand, was the most effective cytokine in
eliciting NO production in nontumorigenic E10 cells, suggesting that
IRFs may play an important role in NO induction in these murine lung
epithelial cells. Deletion constructs in iNOS reporter assays (37, 41)
substantiate the importance of at least one of the IRF binding elements
for the full expression of interferon-
-induced NO production in RAW
264.7 cells. Consistent with this report was the demonstration that NO
induction by interferon-
in pancreatic cells was preceded by
increases in nuclear IRF-1 protein (19). Should the transcriptional
activation of iNOS by interferon-
in murine lung epithelial cells be
similarly mediated by IRF-1, the tumorigenic E9 and A5 epithelial cells
may have a defect in the IRF transduction pathways.
Individual cytokines had varying effects on NO production in the cell
lines. In all cell lines, interleukin-1
failed to elicit NO
production. This contrasts with interleukin-1
-induced increases in
NO from L2 cells (a rat nontumorigenic type II cell line; see Ref. 26)
and from rat adult and fetal primary type II cells (24, 48). The
amounts of induced NO in these rat and human cells were less than those
caused by a mixture of cytokines, however. In A549 cells, a line
derived from human lung carcinoma, exclusion of interleukin-1
from
cytomix plus lipopolysaccharide reduced iNOS gene expression by 41%
(3), indicating a contributory role of this cytokine in NO induction.
An absence of receptors for interleukin-1
on cells used in this
study could be responsible for the failure of this cytokine to affect
NO production in cells; this bears further study. Induction of NO
synthesis by tumor necrosis factor-
appeared to be cell line
selective in that a modest induction of NO synthesis occurred in E10
cells but not in C10, E9, or A5 cells. In other lung epithelial cell
lines, the effects of this cytokine are equivocal. In A549 cells,
exclusion of tumor necrosis factor-
from the cytomix cocktail
decreased NO synthesis by an amount equivalent to that caused by
deleting interleukin-1
(5). On the other hand, tumor necrosis
factor-
did not affect NO production by lung epithelial cells
cultured from the fetal or adult rat (24, 48) or by L2 cells (26). Of
all the cytokines included in the cytomix cocktail, interferon-
was
the most effective by itself. Indeed, interferon-
elicited NO
induction in E10 and A5 cells approaching that induced by cytomix.
These results are in accordance with other studies in lung epithelial
cells wherein interferon-
plays a pivotal role in NO induction
either alone or in combination with other cytokines (3, 26, 48).
In epithelial cell lines derived from human (A549), rat, or mouse
lungs, dexamethasone inhibits cytomix-induced NO production (26, 52,
53). Similar results were obtained in the present study using murine
E10 cells. The inability of dexamethasone treatment to completely
prevent NO production may relate to this glucocorticoid being
administered at the same time as cytokine application. Indeed, many of
the effects of the glucocorticoids require alterations in protein
synthesis (23), a process that usually takes several hours. In support
of this notion is the observation that the concentration and duration
of exposure to dexamethasone can influence NO generation and
interleukin-1
release from rat alveolar macrophages (6). However,
preincubation of E10 cells with dexamethasone for 24 h before cytokine
application did not enhance the inhibitory effect of dexamethasone on
NO induction (P. Kazakoff, personal communication). Accordingly, signal
transduction pathways activated by cytomix in lung epithelial cells
appear to be both glucocorticoid sensitive and insensitive. NO
production in nonpulmonary cell types can also be modulated by
concurrent glucocorticoid treatment (13, 49).
Cytomix enhanced cell proliferation in both E10 and E9 cells by
40-50% over a 24-h period. The influence of NO on cell
proliferation in other systems is equivocal, with both increases (11)
and decreases (4) on extrapulmonary tumor cell proliferation having been reported. The physiological consequences of NO production on lung
epithelial cell growth are currently not known, although transfection
of a human bronchial epithelial cell line (BEAS-2B) with iNOS had no
effect on cell proliferation even though
c-fos expression was stimulated (18).
Cytomix-induced proliferation was prevented by aminoguanidine, an agent
that shows relative selectivity as an inhibitor of iNOS (43),
suggesting a role for NO in mediating the proliferative action of
cytomix. However, other evidence suggests that the relationship between
NO production and the proliferation may be coincidental. Cytomix
administration induced proliferation in E9 cells, which produce little,
if any, NO. Dexamethasone prevented the proliferation but did not
abolish NO production. Accordingly, other actions of aminoguanidine
aside from NOS inhibition may underlie the inhibition of
cytomix-induced proliferation, such as inhibition of polyamine
metabolism (42) and the glycosylation of cell matrix (8).
Spontaneously transformed murine lung epithelial cells produce less NO
in response to cytokine activation than do their nontumorigenic counterparts. Neoplasia may promote
resistance to cytokine-induced NO
synthesis in certain cell types. The
reduced iNOS expression and activity in human gastric (50) and
colorectal tumor tissue (45) relative to normal tissue is consistent
with such a proposal. Deficient NO could encourage tumor cell
proliferation, inhibit differentiation (10), or prevent neoplastic
cells from undergoing apoptosis (64). The complexity of the role of NO
in cancer is illustrated by the observation that NO has both
pro-apoptotic and anti-apoptotic effects (13), the cellular fate
depending on the cell type and level of NO production. A role for
endogenously produced NO in tumor development has been demonstrated in
a mouse model of hepatic metastasis in which induction of iNOS in the tumor cells led to regression of the metastases (65). It is clear that
the capacity of neoplastic cells to produce NO varies from cell type to
cell type. Indeed, the ability of cytomix to elicit NO production from
lung tumor-derived epithelial cell lines (3, 26, 52, 53) as opposed to
the spontaneous transformants used herein, together with elevated NOS
expression or activity in human breast cancer (61), central nervous
system (11), and gynecological cancers (60), argues against deficient
NO being common to all neoplastic cells. The amounts of NO generated by
the tumor-derived cell lines relative to the cells from which they
transformed is unknown. Conceivably, the nontumorigenic cells may
respond with significantly higher levels of NO than their neoplastic
derivatives. This consideration emphasizes the value of the sibling
cell pairs used in the present study. In some human tumors, iNOS
expression was not increased as much as other NOS isoforms (11, 60),
and in breast cancer, iNOS expression was localized to the
tumor-infiltrating macrophages rather than the tumor parenchyma (61).
The ability of neoplastic cells to produce NO and/or respond to
NO may be dictated by factors such as malignancy, differentiation
status (60), and metastatic potential (15, 64). The microenvironment
affects the response of tumor cell lines (EMT-6 murine breast cancer,
DLD-1 human colon adenocarcinoma) in that NO overexpression inhibits
tumor cell growth in vitro but enhances tumor growth in vivo (17, 29).
The role of NO in neoplasia is thus not a simple one and warrants
further investigation. The sibling
lung epithelial cell lines described
herein represent unique systems for examining in molecular detail the
components that regulate cytokine induction of NO.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. A. Robbins for the provision of murine inducible
nitric oxide synthase cDNA.
 |
FOOTNOTES |
This work was supported by seed grants from the University of Colorado
Cancer Center and the American Cancer Society and by National Cancer
Institute Grant CA-33497.
Address for reprint requests: D. C. Thompson, Dept. of Pharmaceutical
Sciences, Univ. of Colorado School of Pharmacy, 4200 East Ninth Ave.,
Denver, CO 80262-0238.
Received 19 December 1997; accepted in final form 24 February
1998.
 |
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