Download Early Loss of Fhit in the Respiratory Tract of

Research Article
Early Loss of Fhit in the Respiratory Tract of Rodents
Exposed to Environmental Cigarette Smoke
1
1
1
Francesco D’Agostini, Alberto Izzotti, Roumen Balansky, Nicola Zanesi,
2
1
Carlo M. Croce, and Silvio De Flora
2
1
Department of Health Sciences, University of Genoa, Genoa, Italy and 2Comprehensive Cancer Center, Ohio State
University, Columbus, Ohio
Abstract
The Fhit gene, encompassing the most active common human
chromosomal fragile region, FRA3B, has been shown to act as
a tumor suppressor. Several studies have shown significant
Fhit alterations or Fhit protein loss in lung cancers from
smokers compared with lung cancers from nonsmokers. To
evaluate the role of Fhit under controlled experimental
conditions, we exposed rodents to environmental cigarette
smoke (ECS) and evaluated Fhit expression or Fhit protein in
the respiratory tract. After 14 days of exposure to ECS, loss of
Fhit protein in the bronchial/bronchiolar epithelium affected
half of the tested B6-129(F1) mice, either wild type or Fhit +/ .
After 28 days, it affected the vast majority of the tested SKH-1
hairless mice and of A/J mice and all (UL53-3 x A/J)F1 mice,
either wild type or P53 +/ . In Sprague-Dawley rats, exposure
to ECS for up to 30 days caused a time-dependent loss of Fhit
in pulmonary alveolar macrophages. Moreover, ECS downregulated Fhit expression and significantly decreased Fhit
protein in the rat bronchial epithelium. The oral administration of N-acetylcysteine attenuated the ECS-related loss of
Fhit, whereas oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate, and indole 3-carbinol, and their combinations had no
significant effect. Parallel studies evaluated a variety of
molecular, biochemical, and cytogenetic alterations in the
respiratory tract of the same animals. In conclusion, there is
unequivocal evidence that Fhit is an early, critical target in
smoke-related lung carcinogenesis in rodents, and that
certain chemopreventive agents can attenuate the occurrence
of this gene alteration. (Cancer Res 2006; 66(7): 3936-41)
Introduction
Ten years ago, the Fhit gene was discovered at the common
fragile site FRA3B (1). Fhit encompasses the carcinogen-sensitive
and viral integration–susceptible fragile region. The first coding
exon 5 was found susceptible to deletion in many tumors, resulting in inactivation of Fhit protein. Thus far, almost 550 reports
on Fhit studies have appeared, and Fhit was shown altered in
a large fraction of tumors, including lung cancer (reviewed in
ref. 2).
Several studies, using either immunohistochemical or molecular
methods, showed a significant loss of Fhit in lung cancers from
Note: R. Balansky is currently at the National Center of Oncology, Sofia 1756,
Bulgaria.
Requests for reprints: Silvio De Flora, Department of Health Sciences, University
of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy. Phone: 39-10-353-8500; Fax: 39-10-3538504; E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-3666
Cancer Res 2006; 66: (7). April 1, 2006
smokers compared with lung cancers from nonsmokers (3–9).
Interestingly, a dose-dependent decrease of Fhit methylation was
observed in bronchoalveolar lavage cells from cancer-free patients
undergoing fiber optic bronchoscopy, as related to the number of
cigarettes smoked in a lifetime (10). A small proportion of
histologically normal bronchial epithelium from smokers displayed
either Fhit loss, as detected by immunohistochemistry (11), or an
increased Fhit methylation (12). On the other hand, negative results
were reported in other studies evaluating the association of Fhit
loss with smoking history (13–17).
Thus, on the whole, the assumption that the Fhit gene is a target
for cigarette smoke is mostly supported by studies evaluating Fhit
alterations or Fhit loss in lung tumors. Moreover, the conclusions
of the studies on this subject are not univocal. As a further
consideration, it is not clear whether the Fhit loss is an early
consequence of exposure to cigarette smoke or the result of the
multiple alterations occurring in preneoplastic or neoplastic cells.
These premises prompted us to evaluate changes in Fhit gene
expression and loss of Fhit protein in the respiratory tract of
rodents exposed to environmental cigarette smoke (ECS) for short
periods of time, up to a maximum of 30 days, before appearance
of any ECS-related histopathologic alteration. In these studies,
we used Sprague-Dawley rats and mice belonging to various
strains and genotypes, including B6-129(F1) mice, either wild type
or Fhit +/ ; SKH-1 hairless mice, A/J mice, and (UL53-3 A/J)F1
mice, either wild type or P53 +/ . Part of these animals have been
used in parallel studies evaluating the occurrence of ECS-related
induction of lung tumors and modulation of intermediate
biomarkers in cells of the respiratory tract (18–23). In addition,
in one of the herein reported studies, we exposed hairless mice not
only to ECS but also to ECS plus light or light alone, which induced
skin tumors in mice (24, 25) as well as molecular and biochemical
alterations not only in skin but, surprisingly, even in the respiratory
tract and bone marrow cells (19, 21).
A further goal of the present study was to evaluate whether the
oral administration of putative cancer chemopreventive agents
may influence the ECS-related Fhit loss in rodent bronchial/
bronchiolar epithelial cells. The investigated agents included
sulindac, a nonsteroidal antiinflammatory drug; 5,6-benzoflavone,
a synthetic flavonoid; the thiol N-acetylcysteine; the dithiolthione
oltipraz; the natural compound phenethyl isothiocyanate,
contained in watercress; and the indole glucosinate indole 3carbinol (I3C), a decomposition product of cruciferous vegetables.
The results obtained show that exposure of rats and mice,
belonging to various strains and genotypes, to ECS, for up to
30 days, consistently causes a significant and time-related decrease
of Fhit gene expression, as detected both by reverse transcriptasePCR (RT-PCR) and quantitative real-time PCR (QPCR), and a loss
of Fhit protein, as detected both by immunohistochemistry
and Western blot. Of the chemopreventive agents tested, only
3936
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.
Cigarette Smoke and Fhit
N-acetylcysteine was successful to attenuate the ECS-induced Fhit
down-regulation and Fhit loss.
Materials and Methods
Animals. A total of 97 adult male Sprague-Dawley rats, weighing 315 to
320 g at the start of the experiments, were purchased from Harlan Italy
(Correzzana, Milan, Italy). A total of 34 adult female B6-129(F1) mice, 14 of
which were wild type and 20 heterozygous for Fhit (Fhit +/ ), weighing on
average 35 g, were produced at the Kimmel Cancer Center (Thomas
Jefferson University, Philadelphia, PA) and shipped to the University of
Genoa. Sixteen adult female SKH-1 hairless mice, weighing 21 to 22 g, and
10 adult female A/J mice, weighing 20 to 21 g, were purchased from
Charles River (Calco, Lecco, Italy). Twenty adult female (UL53-3 A/J) F1
mice, 10 of which were wild type and 10 were P53 transgenic, weighing 17
to 18 g, were produced at the Ohio State University (Columbus, OH) and
kindly supplied by Dr. Ming You (University of Columbus, OH) to the
University of Genoa. All animals were housed in Makrolon cages on
sawdust bedding and maintained on standard rodent chows for mice
(MIL, Morini, S. Polo d’Enza, Italy) and rats (Teklad TRM, Harlan) and tap
water ad libitum. The temperature of the animal room was 23 F 2jC,
with a relative humidity of 55% and a 12-hour day/night cycle. The
housing and treatments of animals were in accordance with our national
and institutional guidelines.
Exposure of animals. A whole-body exposure of rats and mice, for the
times indicated in Results, was achieved by burning Kentucky reference
cigarettes (Tobacco Research Institute, University of Kentucky, Lexington,
KY) in a smoking machine (model TE-10, Teague Enterprises, Davis, CA).
2R1 cigarettes, having a declared content of 44.6 mg tar and 2.5 mg nicotine
each, were used in the study with SKH-1 mice. 1R3 cigarettes, having a
declared content of 22.8 mg tar and 1.5 mg nicotine each, were used in all
other studies. Before use, the cigarettes were kept for 48 hours in a
standardized atmosphere humidified with a mixture of 70% glycerol and
30% water. Each smoldering cigarette was puffed for 2 seconds, once every
minute, for a total of eight puffs, at a flow rate of 1.05 L/min to provide a
standard puff of 35 cm3. The smoking machine was adjusted to burn five
cigarettes at one time and to produce a mixture of sidestream smoke (89%)
and mainstream smoke (11%), mimicking exposure to high-dose ECS.
Exposure to ECS was 6 h/d, divided into two rounds with a 3-hour interval,
for the number of days indicated in Results.
Exposure of SKH-1 mice to light was obtained by using halogen quartz
bulbs (12 V, 50 W), supplied by Leuci (File, Lecco, Italy) and equipped with
UV-C filters (WG 280, Schott Optics Division, Mainz, Germany), at an
illuminance level of 10,000 lx. Eight mice were exposed to the light for 9 h/d.
Sixteen additional mice were exposed to daily cycles of both light and ECS,
and half of them received oral sulindac (19).
Treatment with chemopreventive agents. Six chemopreventive agents
were used, either individually or in combination, for assessing their
influence on Fhit protein loss in ECS-exposed rats and mice. Sulindac and
5,6-benzoflavone were purchased from Sigma Chemical Co. (St. Louis, MO);
N-acetylcysteine was purchased from Zambon Italia (Vicenza, Italy); and
oltipraz, phenethyl isothiocyanate, and I3C were supplied by the Division of
Cancer Prevention Repository of the National Cancer Institute (Rockville,
MD). Sulindac and N-acetylcysteine were given with the drinking water, at a
calculated intake of 45 and 1,000 mg/kg body weight, respectively. The other
agents were incorporated in diets, prepared once per week, containing
the concentrations of 400 mg/kg diet (oltipraz), 500 mg/kg diet
(5,6-benzoflavone and phenethyl isothiocyanate), or 2,500 mg/kg diet
(I3C). Treatment with the chemopreventive agents started 3 days before the
first exposure of rodents to ECS and/or light.
Collection and preparation of pulmonary alveolar macrophages
and lung samples. At the times indicated in Results, the animals were
deeply anesthetized with diethyl ether and killed by cervical dislocation. Rat
pulmonary alveolar macrophages (PAM) were collected by bronchoalveolar
lavage (19) and spun onto slides by means of a cytocentrifuge. The slides
were air-dried and fixed in absolute methanol. The right lung from variously
treated rats was immediately stored at 80jC for molecular analyses. The
www.aacrjournals.org
left lung of each animal was fixed in formalin and routinely processed for
Fhit immunohistochemistry.
RNA extraction and Fhit gene expression. RNA was extracted from
the pooled lung samples of rats, either sham exposed or ECS exposed or
treated with N-acetylcysteine, alone or in combination with exposure to
ECS, by sequential proteinase K-DNase I and phenol/chloroform treatments and isopropanol precipitation, as previously reported (20). Fhit
gene expression was evaluated both by semiquantitative RT-PCR and by
QPCR. Specific primers for Fhit were designed by using a commercially
available software (Primer Premier 4, Premier Biosoft International, Palo
Alto, CA), as follows: 5V-TGCTTGGTCACTTGCTCTGC-3V (P1) and 3V-CCTTTGGGCAACATGGACCG-5V (P2). Complementary DNA was amplified by
plateau-PCR amplification.
For RT-PCR analyses, the reaction product of 184 bp was separated by
agarose gel electrophoresis, staining with ethidium bromide, and identified
by making reference to a DNA ladder (pUC18DpnI Digest, Sigma Chemical).
The cDNA amounts were quantified by densitometric analysis using a
digital acquisition equipment (DC 120 Zoom Digital Camera, Eastman
Kodak, Rochester, NY) and a specifically designed software (1D Image
Analysis Software, Eastman Kodak). The results were standardized by
making reference to the expression of the ubiquitin housekeeping gene,
detected at 231 bp.
For QPCR analyses, a Platinum Taq DNA polymerase (Invitrogen,
Carlsbad, CA) reaction was done in a rotating thermocycler (Rotor-Gene
3000, Corbett Research, Mortlake, Australia), using SYBR GREEN fluorochrome (Invitrogen) as a tracer. Ubiquitin mRNA, as detected by
5V-CCTTGTCCTCCGCCTGAG-3V(P1) and 3V-GTCACTGTGGTAGCTCTTGC5V(P2) primers, was tested as an internal positive standard used for data
normalization. Negative controls (DNA-free samples) were included in each
analysis.
Fhit protein. Fhit protein was detected by immunohistochemistry in the
bronchial/bronchiolar epithelium of rats and mice and in rat PAM. A
commercially available kit (Histomouse-SP kit, Zymed Laboratories,
San Francisco, CA) was used following the manufacturer’s instructions. A
rabbit anti-Fhit polyclonal antibody (ZR44, Zymed Laboratories) was
used for rat bronchial/bronchiolar epithelium and PAM, at a final concentration of 3 Ag/mL. A rabbit anti-Fhit polyclonal antibody, kindly supplied
by Dr. Kay Huebner (Ohio State University Comprehensive Cancer Center,
Columbus, OH), was used for mouse bronchial/bronchiolar epithelium, at
final dilution of 1:2,000. Blind-coded slides were evaluated by two readers,
each one scoring 1,000 cells per slide.
In addition, the Fhit protein was detected in the pooled lung samples
from either sham-exposed or ECS-exposed rats by Western blot. S12
fractions (100 Ag protein per sample) were transferred to acrylamide gel and
subjected to electrophoresis. The gel was blotted to an Immun-blot
polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and labeled
first with a primary rabbit anti-Fhit polyclonal antibody (ZR44, Zymed
Laboratories) and then with a fluorescent secondary antibody labeled with
Cy3 (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).
The signals were detected by fluorescence laser scanning (Scanarray,
Packard Bioscience, Billerica, MA), and the Fhit protein band (17 kDa)
was identified by making reference to a 100- to 10-kDa protein marker
(Bio-Rad). Arbitrary units were calculated by subtracting the background
from the fluorescence yielded by each sample. Ubiquitin protein (35 kDa),
as detected by Western blot using a primary anti-Ubiquitin rabbit polyclonal antibody (Ub FL-76, Santa Cruz Biotechnology, Santa Cruz, CA), was
used as an internal positive standard for data normalization.
Statistical analyses. The differences between experimental groups
were evaluated by Student’s t test for unpaired data when comparing
quantitative data (mean F SE) and by m2 analysis when comparing frequencies. Correlations were evaluated by simple regression analysis by
using the StatView software (Abacus Concept, Berkeley, CA).
Results
Fhit loss in the bronchial/bronchiolar epithelium of smokeexposed mice. Table 1 summarizes the results obtained in four
3937
Cancer Res 2006; 66: (7). April 1, 2006
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.
Cancer Research
Table 1. Loss of Fhit protein in the bronchial/bronchiolar
epithelium of mice exposed to ECS for either 14 days [B6129(F1) strain] or 28 days (all other strains)
Mouse strain
Gene status
Treatment
SHAM (%)
B6-129(F1)
SKH-1
A/J
(UL53-3 A/J)F1
Wild type
Fhit +/
Wild type
Wild type
Wild type
P53 +/
0/7
0/10
0/8
0/5
0/5
0/5
(0)
(0)
(0)
(0)
(0)
(0)
ECS (%)
3/7 (42.9)*
5/10 (50.0)c
7/8 (87.5)b
4/5 (80.0)c
5/5 (100)c
5/5 (100)c
*P V 0.05, compared with sham.
cP V 0.01, compared with sham.
bP V 0.001, compared with sham.
strains of mice exposed to ECS for either 14 days [B16-129(F1)
strain] or 28 days (all other strains). Figure 1 shows examples of
immunohistochemical analyses of two sections of bronchiolar
epithelium from A/J mice, either sham exposed (A) or exposed to
ECS for 28 days (B). Due to the difficulty of calculating the
percentage of cells positive for Fhit protein, which is localized in
the cytoplasm, especially in bronchioli, the results shown in Table 1
are expressed in terms of mice showing an evident loss of Fhit out of
those examined. There was a sharp difference between shamexposed mice and ECS-exposed mice. In fact, all samples from the
40 sham-exposed mice were diffusely positive for Fhit in all cells of
the bronchial/bronchiolar epithelium, similar to that shown in
Fig. 1A. In contrast, the majority of ECS-exposed mice (29 of 40; i.e.,
the 72.5%) displayed a situation similar to that depicted in Fig. 1B.
The Fhit loss affected half of B6-129(F1) mice after just 14 days of
exposure to ECS and was only marginally and not significantly higher
in Fhit+/ mice. After 28 days of exposure, a Fhit loss was observed in
the 87.5% of SKH-1 hairless mice, in the 80.0% of A/J mice, and in the
100% of (UL53-3 A/J)F1 mice, irrespective of the p53 status.
In SKH-1 hairless mice, no loss of Fhit was observed in the
bronchial/bronchiolar epithelium after exposure of the mice to the
light emitted by UV-C-filtered halogen lamps. On the other hand,
all eight mice exposed to daily cycles of light and ECS exhibited
Fhit loss, and this result was unchanged in mice that additionally
received sulindac in drinking water (data not shown).
Fhit protein loss and Fhit gene down-regulation in the
respiratory tract of smoke-exposed rats: effect of chemopreventive agents. Only a tiny proportion of bronchial epithelial
cells from sham-exposed Sprague-Dawley rats, kept in filtered air
for 28 days, did not show the presence of Fhit protein detectable by
immunohistochemistry (Fig. 2). The proportion of Fhit-negative
cells increased 3.5-fold after exposure to ECS for 28 days. Of the
seven chemopreventive treatments tested in ECS-exposed rats, only
N-acetylcysteine, given with the drinking water, was successful to
significantly attenuate the ECS-related loss of Fhit. In contrast,
administration with the diet of oltipraz, phenethyl isothiocyanate,
5,6-benzoflavone, I3C, and the combination of phenethyl isothiocyanate with I3C did not significantly affect the ECS-induced
Fhit loss. The proportion of Fhit-negative cells was lower in rats
receiving the combination of N-acetylcysteine with oltipraz
Cancer Res 2006; 66: (7). April 1, 2006
compared with rats exposed to ECS in the absence of any chemopreventive agent, but this difference was not statistically significant
(Fig. 2).
In addition to immunohistochemical analyses, the levels of Fhit
protein were evaluated by Western blot in lung S12 fractions from
rats, either sham exposed or exposed to ECS. As assessed by testing
two replicates of each sample in two separate experiments, the Fhit
protein underwent a 45% decrease (P < 0.01) following exposure to
ECS for 28 days (Fig. 3A).
The level of Fhit transcription was evaluated by both RT-PCR and
QPCR analyses in the lung of rats, either sham exposed (SHAM),
or smoke exposed (ECS), or receiving oral N-acetylcysteine alone,
or smoke exposed and treated with N-acetylcysteine (ECS +
N-acetylcysteine). RT-PCR (Fig. 3B) resulted in the production of an
amplified sequence of 184 bp corresponding to Fhit mRNA.
As assessed by testing three replicates of each sample in three
separate experiments, N-acetylcysteine alone did not appreciably
decrease Fhit expression, which was 17% lower than in SHAM.
ECS halved Fhit expression ( 51%, P < 0.05), an effect which
was attenuated in rats cotreated with N-acetylcysteine ( 33%
compared with SHAM, P = 0.09 compared with ECS). QPCR
resulted in the amplification of fluorescent products (Fig. 3C). As
Figure 1. Examples of immunohistochemical analysis of two sections of
bronchiolar epithelium from A/J mice, either unexposed (A) or exposed to ECS
for 28 days (B ). The diffuse brownish staining of the cell cytoplasm corresponds
to the presence of the Fhit protein.
3938
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.
Cigarette Smoke and Fhit
reliable to score individual cells rather than cells within tissues.
PAM are sweeping, sentinel cells that can easily be recovered by
bronchoalveolar lavage, which are particularly useful for studying
molecular, cytogenetic, and biochemical alterations in both
humans and animal models (18, 19, 26–28). As many as 1 to 5 106 PAM are removed every hour from human terminal airways via
the mucociliatory escalator (29). The only study in the literature
reporting a cigarette smoke–related effect of Fhit in bronchoalveolar lavage cells from cancer-free smokers is the one by Kim et al.
(10), who showed an increase of Fhit methylation that was dosedependently related to the number of pack-years. In general, the
de novo methylation of CpG islands within the promoters of tumor
suppressor genes is one of the frequent mechanisms of gene
inactivation in lung carcinogenesis (30). Based on the above
findings and taking into account that the half-life of Fhit in PAM
of ECS-exposed rats was <3 months, the possibility of evaluating
Figure 2. Loss of Fhit protein, as assessed by immunohistochemistry in the
bronchial/bronchiolar epithelium of Sprague-Dawley rats exposed to ECS for 28
days and receiving orally chemopreventive agents or their combinations.
Columns, means of the data obtained in the eight rats composing each
experimental group; bars, SE. a, significantly increased (P < 0.001) compared
with sham-exposed rats; b, significantly decreased (P = 0.05) compared with
ECS-exposed rats, in the absence of any chemopreventive agent.
assessed by testing three replicates of each sample, N-acetylcysteine alone did not appreciably decrease Fhit transcription, which
was 13% lower than in SHAM. ECS significantly attenuated Fhit
transcription ( 46%, P < 0.001 compared with SHAM), an effect
that was partially but significantly reverted in rats cotreated with
N-acetylcysteine ( 33% compared with SHAM, P = 0.05 compared
with ECS).
Time course loss of Fhit in PAMs from smoke-exposed rats.
Exposure of Sprague-Dawley rats to ECS for up to 30 days resulted
in a time-dependent loss of Fhit. The proportion of Fhit-positive
PAM was 61.6 F 3.6% at time 0 and 50.3 F 1.0% after 30 days. This
decrease was statistically significant (Fig. 4). Figure 4 also shows
the appearance of two binucleated PAM, one positive (Fhit+) and
one negative (Fhit ) for this protein in immunohistochemistry. The
correlation between Fhit levels in PAM and time of exposure to ECS
(r = 0.994) was statistically significant (P < 0.001). The regression
line is y = 62.05 0.38x, where y is the percentage of Fhit-positive
PAM, and x is the exposure time in days. Based on this equation,
and provided that the loss of Fhit continues to be linear after 30
days of exposure, the half-life of Fhit loss in PAM of ECS-exposed
rats, under our experimental conditions, would be expected to be
81.8 days.
Discussion
The results of the present study provide evidence that a relatively
short-term exposure of rodents to ECS causes a significant downregulation of Fhit gene expression, as assessed by both RT-PCR and
QPCR, as well as a loss of Fhit protein, as assessed by both
immunohistochemistry and Western blot. These effects were
detected in apparently healthy cells of the respiratory tract. In
particular, a time-dependent loss of Fhit was recorded in PAM after
exposure of rats to ECS for varying periods of time. Evaluation of
Fhit by immunohistochemistry in PAM is particularly convenient
because, due to the localization of Fhit in the cytoplasm, it is more
www.aacrjournals.org
Figure 3. Examples of Western blot analysis of Fhit protein (A) and of
RT-PCR (B) and QPCR (C ) analyses of Fhit gene expression in the lung of
Sprague-Dawley rats, either untreated (SHAM) or treated with N -acetylcysteine
(NAC ) or exposed to ECS for 28 days or exposed to ECS and treated with
N -acetylcysteine (ECS + NAC ).
3939
Cancer Res 2006; 66: (7). April 1, 2006
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.
Cancer Research
Figure 4. Time-dependent loss of Fhit protein in PAM of Sprague-Dawley rats
exposed to ECS for up to 30 consecutive days. Points, means of the data
obtained in the five rats composing each experimental group; bars, SE. Two
binucleated PAM, one negative and one positive for Fhit in immunohistochemistry.
a, significantly decreased (P < 0.05) compared with time 0.
Fhit gene or Fhit protein changes in PAM as a marker of exposure
to cigarette smoke or other inhalant carcinogens and, perhaps, as a
tool for distinguishing susceptible smokers requires further studies.
In addition, loss of Fhit protein in the bronchial/bronchiolar
epithelium affected about half of the B6-129(F1) mice exposed to
ECS for 14 days only, irrespective of the Fhit status. This result
may be in accordance with a previous experiment in which wildtype and Fhit +/ mice treated with the pulmonary carcinogen
4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone did not exhibit significantly different frequencies of lung lesions (31). The vast
majority of the tested SKH-1 hairless mice and of A/J mice and all
(UL53-3 A/J)F1 mice, either wild type or with a germ line
inactivation of one allele of P53, which is a hallmark of Li-Fraumeni
syndrome (32), displayed a loss of Fhit after 28 days of exposure to
ECS. It is noteworthy that Fhit and P53 work synergistically as
tumor suppressors and Fhit-mediated inactivation of mdm2 leads
to P53 stabilization (33). Our findings document, under well
controlled experimental conditions, that Fhit is indeed a critical
target for cigarette smoke, and that this molecular change occurs
early in the carcinogenesis process and not as a consequence of the
development of the neoplastic mass. This is accordingly to a
number of previous studies (reviewed in ref. 34). The decreased
transcription of Fhit in ECS-exposed rats suggests that Fhit loss is
the result of genomic changes, such as deletion or methylation, and
not of postgenomic alterations, such as production of mutated
protein or posttranscriptional regulation.
On this ground, prevention strategies can be aimed either at
inhibiting Fhit alterations or at restoring the damaged gene. The
latter approach has already been pursued by oral gene transfer,
using adenoviral or adeno-associated viral vectors expressing the
human Fhit gene in heterozygous Fhit +/ knockout mice (35, 36).
The cDNA sequence and structure of murine Fhit are similar to
those of the human gene, with exons 5 to 9 encoding the protein
(37). The other strategy to prevent Fhit inactivation is to use
dietary and/or pharmacologic agents aimed at down-regulating the
mechanisms that trigger Fhit alterations. Of the chemopreventive
agents evaluated in the present study, sulindac failed to attenuate
the loss of Fhit protein in the bronchial/bronchiolar epithelium
of SKH-1 mice. In Sprague-Dawley rats, out of seven chemopreventive regimens tested, only administration of N-acetylcysteine in
Cancer Res 2006; 66: (7). April 1, 2006
drinking water was effective in attenuating both the ECS-induced
down-regulation of Fhit gene expression in lung and Fhit protein
loss in the bronchial/bronchiolar epithelium. In contrast, oltipraz,
phenethyl isothiocyanate, 5,6-benzoflavone, I3C, and a combination of phenethyl isothiocyanate with I3C did not significantly
affect the Fhit loss. With the exception of oltipraz, all above
agents have been found to inhibit by at least 50% the formation of
ECS-induced bulky DNA adducts in the lung of the same animals
and had various effects when we investigated, by cDNA microarrays, their ability to modulate the expression of 4,858 genes in
lung and liver of both unexposed and ECS-exposed rats (22). In
addition, when studying by antibody microarray the levels of 518
proteins in the lung of the same animals, N-acetylcysteine did not
change per se the level of any tested protein but decreased the
number of ECS-induced proteins (38).
N-acetylcysteine is an analogue and precursor of L-cysteine and
reduced glutathione, which has been shown to exert a variety of
protective effects and mechanisms in mutagenesis and carcinogenesis (39, 40). Interestingly, in the same rats used in the present
study, N-acetylcysteine and its combination with oltipraz were the
only treatments capable of significantly decreasing the frequency
of apoptotic cells in the bronchial/bronchiolar epithelium. In
contrast, phenethyl isothiocyanate showed an opposite trend, and
all other treatments had no significant effect (23). These data are in
line with the results of other studies showing a decrease by
N-acetylcysteine and an increase by phenethyl isothiocyanate
of apoptotic PAM in rats exposed to ECS, and a decrease by
N-acetylcysteine of apoptotic cells in the bronchial/bronchiolar
epithelium of rats exposed to mainstream cigarette smoke (23).
Enhancement of apoptosis is conceptually a double-edged sword,
because it provides a protective mechanism in carcinogenesis but
may contribute to the pathogenesis of other degenerative diseases.
On the other hand, inhibition of apoptosis by chemopreventive
agents, as observed with N-acetylcysteine, is expected to reflect
their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress
that trigger apoptosis (23). The circumstance that N-acetylcysteine
was the only agent able to attenuate both apoptosis and Fhit
protein loss in the respiratory tract of ECS-exposed rats is
meaningful, because it suggests that Fhit alterations and the
apoptotic process are triggered by the same or parallel mechanisms. In this regard, it is noteworthy that Fhit has been involved
in the regulation of apoptosis (41). In terms of mechanisms,
previous studies done on lung tumors from mice treated with the
carcinogen vinyl carbamate provided evidence that loss of Fhit
expression and promoter/exon 1/intron 1 methylation status were
correlated (42).
In conclusion, the herein reported data support and elucidate
the role of Fhit as an early target in cigarette smoke–related lung
carcinogenesis and indicate that certain chemopreventive agents
can attenuate the induction of this gene alteration.
Acknowledgments
Received 10/12/2005; revised 1/18/2006; accepted 2/3/2006.
Grant support: Associazione Italiana per la Ricerca sul Cancro, NIH National
Cancer Institute, and Bulgarian Ministry of Education and Science.
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.
We thank Dr. Ming You for providing the (UL53-3 A/J)F1 mice and Drs. Maria
Bagnasco, Cristina Cartiglia, Mariagrazia Longobardi, and Maricel Rocha for their
skilful technical assistance.
3940
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.
Cigarette Smoke and Fhit
References
1. Ohta M, Inoue H, Cotticelli MG, et al. The FHIT gene,
spanning the chromosome 3p14.2 fragile site and renal
carcinoma-associated t(3;8) breakpoint, is abnormal in
digestive tract cancers. Cell 1996;84:587–97.
2. Huebner K, Croce CM. Cancer and the FRA3B/FHIT
fragile locus: it’s a HIT. Br J Cancer 2003;88:1501–6.
3. Sozzi G, Sard L, De Gregorio L, et al. Association
between cigarette smoking and FHIT gene alterations in
lung cancer. Cancer Res 1997;57:2121–3.
4. Sozzi G, Pastorino U, Moiraghi L, et al. Loss of FHIT
function in lung cancer and preinvasive bronchial
lesions. Cancer Res 1998;58:5032–7.
5. Marchetti A, Pellegrini S, Sozzi G, et al. Genetic
analysis of lung tumours of non-smoking subjects: p53
gene mutations are constantly associated with loss of
heterozygosity at the FHIT locus. Br J Cancer 1998;78:
73–8.
6. Tomizawa Y, Nakajima T, Kohno T, Saito R, Yamaguchi
N, Yokota J. Clinicopathological significance of Fhit
protein expression in stage I non-small cell lung
carcinoma. Cancer Res 1998;58:5478–83.
7. Stein CK, Glover TW, Palmer JL, Glisson BS. Direct
correlation between FRA3B expression and cigarette
smoking. Genes Chromosomes Cancer 2002;34:333–40.
8. Chang YL, Wu CT, Shih JY, Lee YC. Roles of Fhit and
p53 in Taiwanese surgically treated non-small-cell lung
cancers. Br J Cancer 2003;89:320–6.
9. Kim JS, Kim H, Shim YM, Han J, Park J, Kim DH.
Aberrant methylation of the FHIT gene in chronic
smokers with early stage squamous cell carcinoma of
the lung. Carcinogenesis 2004;25:2165–71.
10. Kim H, Kwon YM, Kim JS, et al. Tumor-specific
methylation in bronchial lavage for the early detection
of non-small-cell lung cancer. J Clin Oncol 2004;22:
2363–70.
11. Tseng JE, Kemp BL, Khuri FR, et al. Loss of Fhit
is frequent in stage I non-small cell lung cancer and
in the lungs of chronic smokers. Cancer Res 1999;59:
4798–803.
12. Zochbauer-Muller S, Fong KM, Maitra A, et al. 5VCpG
island methylation of the FHIT gene is correlated with
loss of gene expression in lung and breast cancer.
Cancer Res 2001;61:3581–5.
13. Nelson HH, Wiencke JK, Gunn L, Wain JC, Christiani
DC, Kelsey KT. Chromosome 3p14 alterations in lung
cancer: evidence that FHIT exon deletion is a target of
tobacco carcinogens and asbestos. Cancer Res 1998;58:
1804–7.
14. Zienolddiny S, Ryberg D, Arab MO, Skaug V, Haugen
A. Loss of heterozygosity is related to p53 mutations and
smoking in lung cancer. Br J Cancer 2001;84:226–31.
www.aacrjournals.org
15. Wistuba II, Behrens C, Virmani AK, et al. High
resolution chromosome 3p allelotyping of human lung
cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele
loss and three regions of frequent breakpoints. Cancer
Res 2000;60:1949–60.
16. Pylkkanen L, Wolff H, Stjernvall T, et al. Reduced Fhit
protein expression and loss of heterozygosity at FHIT
gene in tumours from smoking and asbestos-exposed
lung cancer patients. Int J Oncol 2002;20:285–90.
17. Tomizawa Y, Iijima H, Nomoto T, et al. Clinicopathological significance of aberrant methylation of RARbeta2 at 3p24, RASSF1A at 3p21.3, and FHIT at 3p14.2 in
patients with non-small cell lung cancer. Lung Cancer
2004;46:305–12.
18. De Flora S, Balansky RM, D’Agostini F, et al.
Molecular alterations and lung tumors in p53 mutant
mice exposed to cigarette smoke. Cancer Res 2003;63:
793–800.
19. Balansky RM, Izzotti A, D’Agostini F, et al. Systemic
genotoxic effects produced by light, and synergism with
cigarette smoke in the respiratory tract of hairless mice.
Carcinogenesis 2003;24:1525–32.
20. Izzotti A, Cartiglia C, Longobardi M, et al. Gene
expression in the lung of p53 mutant mice exposed to
cigarette smoke. Cancer Res 2004;64:8566–72.
21. Izzotti A, Cartiglia C, Longobardi M, et al. Alterations
of gene expression in skin and lung of mice exposed to
light and cigarette smoke. FASEB J 2004;18:1559–61.
22. Izzotti A, Bagnasco M, Cartiglia C, et al. Modulation
of multigene expression and proteome profiles by
chemopreventive agents. Mutat Res 2005;591:212–23.
23. D’Agostini F, Izzotti A, Balansky RM, Bennicelli C, De
Flora S. Modulation of apoptosis by chemopreventive
agents. Mutat Res 2005;591:173–86.
24. De Flora S, D’Agostini F. Halogen lamp carcinogenicity. Nature 1992;356:569.
25. D’Agostini F, De Flora S. Potent carcinogenicity of
uncovered halogen lamps. Cancer Res 1994;54:5081–5.
26. De Flora S, Izzotti A, D’Agostini F, Rossi GA, Balansky
R. Pulmonary alveolar macrophages in molecular
epidemiology and chemoprevention of cancer. Environ
Health Perspect 1993;99:249–52.
27. Balansky R, D’Agostini F, De Flora S. Induction,
persistence and modulation of cytogenetic alterations in
cells of smoke-exposed mice. Carcinogenesis 1999;20:
1491–7.
28. Izzotti A, Balansky RM, D’Agostini F, et al. Modulation
of biomarkers by chemopreventive agents in smokeexposed rats. Cancer Res 2001;61:2472–9.
29. Green GM, Jakab GJ, Low RB, Davis GS. Defense
mechanisms of the respiratory membrane. Am Rev
Respir Dis 1977;115:479–514.
3941
30. Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP.
Alterations in DNA methylation: a fundamental aspect
of neoplasia. Adv Cancer Res 1998;72:141–96.
31. Zanesi N, Mancini R, Sevignani C, et al. Lung cancer
susceptibility in Fhit-deficient mice is increased by Vhl
haploinsufficiency. Cancer Res 2005;65:6576–82.
32. Zhang Z, Liu Q, Lantry LE, et al. A germ-line p53
mutation accelerates pulmonary tumorigenesis: p53independent efficacy of chemopreventive agents green
tea or dexamethasone/myo-inositol and chemotherapeutic agents taxol or Adriamycin. Cancer Res 2000;60:
901–7.
33. Nishizaki M, Sasaki J-i, Fang B, et al. Synergistic
tumor suppression by coexpression of FHIT and p53
coincides with FHIT-mediated MDM2 inactivation and
p53 stabilization in human non-small cell lung cancer
cells. Cancer Res 2004;64:5745–52.
34. Ishii H, Ozawa K, Furukawa Y. Alterations of the
fragile histidine triad gene early in carcinogenesis: an
update. J Exp Ther Oncol 2003;3:291–6.
35. Dumon KR, Ishii H, Fong LY, et al. FHIT gene therapy
prevents tumor development in Fhit-deficient mice.
Proc Natl Acad Sci U S A 2001;98:3346–51.
36. Zanesi N, Pekarsky Y, Croce CM. A mouse model of
the fragile gene FHIT: from carcinogenesis to gene
therapy and cancer prevention. Mutat Res 2005;591:
103–9.
37. Pekarsky Y, Druck T, Cotticelli MG, et al. The murine
Fhit locus: isolation, characterization, and expression in
normal and tumor cells. Cancer Res 1998;58:3401–8.
38. Izzotti A, Bagnasco M, Cartiglia C, et al. Chemoprevention of genome, transcriptome, and proteome
alterations induced by cigarette smoke in rat lung. Eur J
Cancer 2005;41:1864–74.
39. De Flora S, Izzotti A, D’Agostini F, Balansky RM.
Mechanisms of N -acetylcysteine in the prevention of
DNA damage and cancer, with special reference to
smoking-related end-points. Carcinogenesis 2001;22:
999–1013.
40. De Flora S, Izzotti A, Albini A, D’Agostini F, Bagnasco
M, Balansky RM. Antigenotoxic and cancer preventive
mechanisms of N -acetyl-L-cysteine. In: Kelloff GJ, Hawk
ET, Sigman CC, editors. Cancer chemoprevention. Vol. 1.
Promising cancer chemopreventive agents. Totowa (NJ):
The Humana Press Inc.; 2004. p. 37–67.
41. Sard L, Accornero P, Tornielli S, et al. The tumorsuppressor gene FHIT is involved in the regulation of
apoptosis and in cell cycle control. Proc Natl Acad Sci
U S A 1999;96:8489–92.
42. Han S-Y, Iliopoulos D, Druck T, et al. CpG
methylation in the Fhit regulatory region: relation to
Fhit expression in murine tumors. Oncogene 2004;23:
3990–8.
Cancer Res 2006; 66: (7). April 1, 2006
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.
Early Loss of Fhit in the Respiratory Tract of Rodents
Exposed to Environmental Cigarette Smoke
Francesco D'Agostini, Alberto Izzotti, Roumen Balansky, et al.
Cancer Res 2006;66:3936-3941.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/66/7/3936
This article cites 41 articles, 23 of which you can access for free at:
http://cancerres.aacrjournals.org/content/66/7/3936.full.html#ref-list-1
This article has been cited by 5 HighWire-hosted articles. Access the articles at:
/content/66/7/3936.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2006 American Association for Cancer
Research.