Affiliations of authors: S. P. Shriver, H. A. Bourdeau, C. T. Gubish, D. L. Tirpak, A. L. G. Davis, J. M. Siegfried (Department of Pharmacology), J. D. Luketich (Department of Surgery), Lung Cancer Program, University of Pittsburgh Cancer Institute, University of Pittsburgh, PA.
Correspondence to present address: Sharon Persinger Shriver, Ph.D., Department of Biology, The Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802-5301 (e-mail: sps10{at}psu.edu).
Present address: D. L. Tirpak, Graduate School of Public Health, University of Texas Health Science Center, Houston.
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ABSTRACT |
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INTRODUCTION |
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Growth stimulation by bombesin-like peptides has been shown to play an important role in human carcinogenesis. GRPRs and/or bombesin-dependent growth stimulation have been observed in human prostate, breast, and gastric carcinomas (7-13). In the case of lung cancer, GRP has been well documented as an autocrine growth factor for both small-cell lung cancer and non-small-cell lung cancer (NSCLC), and bombesin-like peptides can promote proliferation of lung tumor cell lines (3,14). Expression of messenger RNA (mRNA) for the three bombesin-like peptide receptors has been observed in NSCLC, and it has been shown that transfer of the GRPR gene to immortalized bronchial epithelial cells confers an increased growth response to GRP (15). Elevated levels of GRP have been observed in the urine of asymptomatic cigarette smokers compared with normal nonsmokers [P = .007; (16)]. Expression of mRNA for GRPR is also associated with long-term smoking (3). These studies suggest that bombesin-like peptides and their receptors play a role in the promotion of lung carcinogenesis.
Important differences in susceptibility to lung cancer exist between men and women (17-20). The risk for all major lung cancer types is consistently higher in women than in men at every level of exposure to cigarette smoke; odds ratios for an association of lung cancer with smoking are 1.2-fold to 1.7-fold higher for women than for men, depending on the histologic type of lung cancer (17). Factors such as differences in baseline exposure, smoking history, or body size do not account for the increased risk, which is likely due to a higher susceptibility to the effects of tobacco carcinogens in women (17-20). The airways of females also exhibit a higher degree of bronchial responsiveness to cigarette smoke compared with those of males of all age groups, and airways of females appear more susceptible to adverse effects of cigarette smoke than those of males (21). The gene encoding the GRPR, which mediates the proliferative effects of bombesin-like peptides in the lung (3), is located on the X chromosome and has been shown to escape X inactivation in somatic cell hybrids produced from the fibroblasts and lymphoblasts of normal women (22). The GRPR gene is located on the distal end of the p arm of the X chromosome, adjacent to the pseudoautosomal region, which is known to contain clusters of genes that escape X inactivation. Thus, women may have two actively transcribed alleles of the GRPR gene, compared with one in men.
We hypothesized that the additional active copy of the GRPR gene in women may result in more frequent expression of GRPR mRNA in nonsmoking women and/or an increased frequency of activation of GRPR expression in female smokers. In this study, we have examined GRPR mRNA expression in cultured normal airway cells and tissues from male and female subjects with various smoking histories. As a control, we have also examined mRNA expression of the gene encoding the related receptor, neuromedin B receptor. The neuromedin B receptor was found previously not to be associated with smoking or proliferative response to bombesin-like peptides (3) and is located on an autosome [chromosome 6; (23)], in the same group of subjects.
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SUBJECTS AND METHODS |
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Experiments were conducted with the use of tissues and cultured cells from 78 individuals
(Table 1). Tissues were collected under an approved institutional review
board protocol. Biopsy specimens were obtained sequentially from patients who agreed to
participate in the study and who signed an informed consent form. Included were 71 lung
resection or bronchoscopy patients, five lung transplantation patients, and two normal lung
donors. Of the 71 resection or bronchoscopy patients, 58 were diagnosed with lung carcinoma
(including 26 with adenocarcinoma, 20 with squamous cell carcinoma, three with bronchoalveolar
carcinoma, two with large-cell carcinoma, one with malignant carcinoid tumor, and six with
undifferentiated or mixed cell carcinomas), five with a benign lung tumor, three with carcinoma
from a distant organ that metastasized to the lung, and three with emphysema or chronic
obstructive pulmonary disease. One resection patient was diagnosed with lymphoma, and another
was diagnosed with a squamous cell carcinoma of the head and neck. Of the five lung transplant
recipients, three had cystic fibrosis, one had pulmonary hypertension, and one had scleroderma.
Medical histories were reviewed for information on diagnosis, age, sex, and smoking history. The
exposure to smoking was estimated as none (for lifetime nonsmokers), short-term (1-25
pack-years [pack-years = number of packs of cigarettes smoked per day multiplied
by the number of years of smoking]), or long-term (>25 pack-years).
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Biopsy specimens of the mainstem bronchus and peripheral lung were obtained at the time of resection, bronchoscopy, lung transplantation, or lung donation. Airway and peripheral lung biopsy specimens did not show pathologic changes. Primary cultures of human bronchial epithelial cells and lung fibroblasts were established from the biopsy tissues in media selective for each cell type as previously described (3). Cells were grown in short-term culture and assayed for GRPR expression at passage 1, within 6 weeks of tissue donation. In several cases, a portion of the peripheral lung or bronchus was snap-frozen in liquid nitrogen at the time of tissue procurement. For some patients, a blood specimen was also obtained, and the peripheral blood lymphocytes (PBLs) were isolated and viably frozen for future analysis.
Nucleic Acid Isolation and Analysis
RNA was isolated from snap-frozen lung tissue, cultured cells, and PBLs by the acid phenol method (24). DNA was isolated for polymerase chain reaction (PCR) from cells with the use of standard methods (25). PCR amplification was done in a reaction volume of 25 µL with the use of 50-200 ng of template, 10 pmol of each primer (one primer end-labeled with 32P; Amersham Life Science Inc., Arlington Heights, IL), 200 µM each deoxynucleotide triphosphate, 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.25 U Taq polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN).
Amplification of the GRPR mRNA following oligo-dT-primed reverse transcription (RT) of total RNA was performed with primers GRPR-1 (5'-CTCCCCGTGAACGATGACTGG) and GRPR-2 (5'-ATCTTCATCAGGGCATGGGAG) for 35 cycles (of 94 °C for 30 seconds, 65 °C for 30 seconds, and 72 °C for 30 seconds). These primers result in a product of 389 base pairs (bp) and are designed to span an intron-exon boundary so that trace amounts of contaminating genomic DNA cannot be amplified under the PCR conditions used.
Southern blots of the RT-polymerase chain reaction (RT-PCR) products were hybridized with a 32P-labeled internal probe (GRPR-D: 5'-CACCTCCATGCTCCACTTTGTC). Primers, PCR conditions, and probe for the amplification and detection of neuromedin B receptor mRNA were as previously described (3).
The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was amplified as a positive control for RT-PCR with the use of primers GAP-1 (5'-GTCAACGGATTTGGTCTGTATT) and GAP-2 (AGTCTTCTGGGTGGCAGTGAT) for 25 cycles (of 94 °C for 1 minute, 58 °C for 1 minute, and 72 °C for 1 minute). Primers are designed to span an intron-exon boundary so that trace amounts of contaminating genomic DNA cannot be amplified under the PCR conditions used. The GAPDH PCR product (520 bp) was detected by Southern blot hybridization with a gene-specific oligonucleotide probe (GAP-3: 5'-CGATGCTGGCGCTGAGTACAT).
RT-PCR assays for GAPDH, GRPR, and neuromedin B receptor were performed in parallel
with the use of the same reagents and RT reactions, and only RNA samples for which a GAPDH
product was obtained were included in the data analysis. Use of GAPDH as a positive control for
RT-PCR ensured that mRNA was of sufficient quality (size and purity) to be reverse transcribed
and amplified by PCR. Representative results for GAPDH and GRPR are shown in Fig. 1. RNA samples for which no GRPR product was visible in autoradiographs
following Southern blotting were considered negative for GRPR mRNA expression. To verify
that no GRPR band was present in negative samples, we carried out densitometry. For all RNA
samples for which no band was visible (e.g., RNA samples from subjects 839 B, 845 B, 855 B,
860 B, and 881 B in Fig. 1
), no absorbance peak was detected by
densitometry, and the absorbance value found by densitometry was less than or equal to
background (an empty lane on the autoradiograph film). We also verified that no mRNA for
GRPR was present in negative samples by carrying out a second round of PCR or by using more
template in a second reaction to show that further cycles of amplification or more starting RNA
would not produce a product. Because the detection method used relies on both amplification of
complementary DNA (cDNA) reverse transcribed from RNA and Southern blotting, it is difficult
to compare results from different blots run at separate times with the use of different probe
preparations for detection. We, therefore, did not attempt to compare quantitatively the intensity
of signals from positive reactions, but instead we classified subjects as either negative
(nonexpressors) or positive (expressors), as in a previous publication (3).
We did, however, notice that a subset of individuals displayed weak signals for GRPR, but not for
GAPDH, compared with the majority of positive subjects. This subset (about 15% of the
subjects) was made up of seven males and five females, the majority of whom had lower smoking
exposures than the mean (see "Discussion" section).
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A polymorphism involving two nucleotides in exon 2 of the GRPR gene was detected in DNA isolated from fresh lung tissue and from cultured cells with the use of allele-specific oligonucleotide hybridization as described (26). The same method was used to detect the exon 2 polymorphism in GRPR mRNA, with the use of the primers GRPR-5 (5'-CAGATGGCTATTTGGCAGGATTG) and GRPR-6 (5'-CCACGGGAAGATTGTAAGCACTC) to amplify oligo-dT-primed total RNA, using an annealing temperature of 64 °C. These primers result in a product of 428 bp and span intron I of the GRPR gene so that genomic DNA cannot be amplified. The RNA polymorphism was detected with the use of the hybridization conditions and probes described by Heidary et al. (26).
Nicotine-Binding Studies
Nicotine-binding experiments were performed as described (27) with
the use of radiolabeled [N-methyl-3H]nicotine
(specific activity, 81.5 Ci/mmol; New England Nuclear, Boston, MA). Briefly, lung fibroblasts or
IB3-1 cells [immortalized human bronchial epithelial cells; (28)] were seeded into 24-well tissue culture plates at a density of 1 x 105
cells per well. Normal, nonimmortalized human bronchial epithelial cells were plated at a density
of 1 x 106 cells per 25-cm3 tissue culture flask. Cells were
exposed in quadruplicate at 4 °C to increasing concentrations of [N-methyl-3H]nicotine (5-150 nM) to determine total binding. For
the assessment of nonspecific binding, 10 µM unlabeled nicotine was added.
Specific binding of [N-methyl-3H]nicotine was
calculated by subtracting the mean nonspecific binding from the mean total binding (see
Fig. 2). In competition experiments (see Fig. 3
), binding of [N-methyl-3H]nicotine was
assessed in quadruplicate in the presence or absence of 0.1 nM or 1.0 nM
unlabeled nicotine, tetraethylammonium chloride, (+)-tubocurarine, or hexamethonium. Nicotine
was obtained from Sigma Chemical Co. (St. Louis, MO); other reagents were obtained from
Research Biochemical International (Natick, MA).
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Normal human lung fibroblasts were exposed to 0.1 µM or 1.0 µM nicotine in the culture medium for 1 day or 5 days. These concentrations reflect the tissue
and serum levels of nicotine in the average smoker (29-31). Control cells
received culture medium without nicotine. Cells were frozen in guanidinium buffer for RNA
extraction at various time points after addition of nicotine. RT-PCR amplification and Southern
blot hybridization using primers for GRPR mRNA and GAPDH mRNA were performed for each
sample as described above. After blotting, hybridization, and exposure to X-ray film, the
autoradiographs were scanned with the use of a densitometer, and the results are expressed as
relative densitometric units (GRPR expression/GAPDH expression) for each sample (see
Fig. 4).
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The following statistical tests were used where appropriate: Fisher's exact test
(comparing frequencies of GRPR expression in Table 2), two-tailed
Student's t test (comparing mean pack-years in Table 3
),
two-tailed Mann-Whitney test (comparing median pack-years in Table 3
),
and analysis of variance (comparing extent of binding of nicotine in Fig. 3
).
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RESULTS |
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A representative Southern blot showing subjects who were positive and negative for GRPR
mRNA (389-bp product) is shown in Fig. 1, along with uniform positive
results for GAPDH mRNA expression (520-bp product). In Fig. 1
, both
female subjects and one of six male subjects are positive for GRPR mRNA expression. The results
of the analysis of all subjects from Table 1
are summarized in Table 2
. By use of RT-PCR, GRPR mRNA expression was detected in 25 of 51
bronchial epithelial cell cultures, 16 of 20 fibroblast cultures, and eight of 14 specimens of
peripheral lung (which included both epithelial and fibroblast cell types). These results are, to our
knowledge, the first documentation of GRPR mRNA expression in human lung fibroblasts,
although both fibroblasts and epithelial cells have been previously shown to respond to GRP (32). In seven patients, more than one specimen type was available for
analysis, and GRPR mRNA expression was consistent in all instances for each subject between
bronchial epithelial cells and lung fibroblasts or between lung fibroblasts and peripheral lung. In
five patients, two different specimen types were positive; in two other patients, two different
specimen types were negative. These data show that it is valid to combine results from different
cell types to compare GRPR mRNA expression in a series of subjects.
As we reported previously (3), the overall frequency of GRPR gene
expression in airway cells and tissues increased with higher exposure to tobacco smoke (Table 2). For all subjects combined, the prevalence of GRPR-positive subjects
was 37.5% (six of 16 subjects) in lifetime nonsmokers, 44.4% (four of nine subjects)
in short-term (1-25 pack-years) smokers, 72.2% (13 of 18 subjects) in smokers in the
exposure category of 26-49 pack-years, and 62.9% (22 of 35 subjects) in smokers with 50
or more pack-years of exposure. A comparison of lifetime nonsmokers and short-term smokers
with long-term smokers (>25 pack-years) revealed that there was a significant difference in
frequency: 10 (40.0%) of 25 positive compared with 35 (66.0%) of 53 positive; P = .048. Chi-squared analysis showed a trend in the data for increasing expression
frequency as smoking exposure increased (P = .06). However, the impact of
smoking is largely due to smoking effects in males compared with females, since females had a
high frequency of expression in the absence of long-term smoking. Chi-squared test for trend as
smoking increased showed statistical significance when males were analyzed separately (P = .012) but not when females were analyzed separately (P = .44).
We observed a large difference in the frequency of GRPR mRNA expression in the lung
between females and males with limited or no smoking history. None of five male nonsmokers
expressed GRPR mRNA, whereas six of 11 female nonsmokers were positive for expression (P = .09, Fisher's exact test; Table 2). A significant
difference in frequency of mRNA expression was also observed between all females and males
with a pack-year history of 25 years or fewer (including nonsmokers): One of 10 males expressed
GRPR mRNA versus nine of 15 females (P = .018, Fisher's exact test). As
pack-years of smoking increased, this difference between female and male smokers disappeared:
Males and females with a smoking exposure greater than 25 pack-years showed no difference in
the frequency of GRPR mRNA expression (19 of 30 males expressed the gene versus 16 of 23
females; P = .43, Fisher's exact test).
These results suggest that the GRPR gene is expressed more frequently in women in the absence of smoking and/or that expression is activated at an earlier time point in response to tobacco exposure in women than in men. Since data on environmental tobacco exposure were not available for these subjects, it is possible that passive smoke exposure affected the frequency of GRPR gene expression in nonsmokers and short-term smokers. Passive smoke exposure could influence the frequency of GRPR mRNA expression in females more than in males, since the possibility that nonsmoking females have a smoking spouse is higher than that for nonsmoking males. Effects on lung cancer risk of passive smoking from a spouse are relatively weak. Passive smoke from a spouse results in an increase in odds ratios for the risk of developing lung cancer to 1.0-1.3 compared with 1.0 for lifetime nonsmokers not exposed to passive smoke from a spouse (33-35), equivalent to fewer than 5 pack-years of active smoking. If GRPR-positive expression in the nonsmoking women in our study were due only to passive smoke, this would still place these nonsmoking subjects in the short-term smoking risk group and would not change the statistical analysis or the gender effect. However, it would mean that women, rather than showing increased frequency of GRPR gene expression in the absence of smoking, show heightened expression in response to tobacco smoke, compared with males.
A comparison of active smoking history with GRPR expression status in men and women
shows that females, on average, express GRPR mRNA after significantly less exposure to tobacco
smoke than males (mean, 56.3 pack-years for males versus 37.4 pack-years for females [P = .037, Student's t test]; median, 50 pack-years for males
versus 42 pack-years for females [P = .05, Mann-Whitney nonparametric
test]; Table 3). No significant difference in pack-years of smoking
was observed between males and females negative for GRPR mRNA expression (mean, 42.6
pack-years in males versus 28.5 pack-years in females [P = .28,
Student's t test]; median, 35 pack-years for males versus 30 pack-years for
females [P = .32, Mann-Whitney nonparametric test]).
Our data are also consistent with the increased susceptibility of females to lung cancer
compared with males that has been reported in larger epidemiologic studies. An analysis of the
pack-years of tobacco smoke exposure at the time of diagnosis in male and female lung cancer
patients in our study demonstrated that the female subjects in our study developed lung cancer
with significantly less tobacco exposure than did the males, as has been shown previously (17-20). The females with lung cancer among our subjects had a mean
smoking history of 41.2 pack-years, compared with 59.9 pack-years for the males (P
= .032), and the median exposure in pack-years was 50 for males and 42 for females (P = .05), whereas age at diagnosis showed no significant difference between males
and females (Table 3). In addition, almost all of the nonsmokers in our
study group who were diagnosed with lung cancer were female (six of 29 female lung cancer
patients were nonsmokers, compared with one of 29 males; P = .05,
Fisher's exact test). It has been previously observed that nonsmokers with lung cancer are
two to three times more likely to be female than to be male (17). Of the
11 female nonsmoking subjects in our study, six expressed GRPR mRNA. Of these six
nonsmoking females positive for GRPR expression, five were diagnosed with lung cancer. Only
one of five nonsmoking females who did not express the GRPR gene in the airway was diagnosed
with lung cancer. Among the 15 female nonsmokers and short-term smokers, there was a
statistically significant association between expressing GRPR mRNA and being diagnosed with
lung cancer (seven of nine females with lung cancer were GRPR mRNA positive in contrast to
one of six females without lung cancer; P = .04, Fisher's exact test). This
association was not observed in males, since male nonsmokers and short-term smokers did not
express GRPR mRNA.
Our data also show that all female lung cancer patients who express GRPR mRNA had a
significantly lower mean and median pack-year tobacco exposure at diagnosis than men with lung
cancer (both those who express and those who do not express GRPR; Table 3). Again, these results suggest that expression of GRPR mRNA in women is
associated with their increased risk of lung cancer (mean, 39.4 pack-years for women versus 62.7
pack-years for men; P = .026 [GRPR positive and GRPR negative];
Table 3
). GRPR-positive women with lung cancer had a median of 44
pack-years of exposure compared with a median of 52 pack-years in GRPR-positive men with
lung cancer (P = .03). Among all lung cancer patients, the group with the lowest
mean tobacco exposure was women who expressed GRPR mRNA in their lung (39.4 pack-years;
Table 3
). In view of the large range of tobacco exposure in the 29 female
lung cancer patients in this study (0-110 pack-years), we did not observe that the lower mean
smoking history of GRPR-expressing women with lung cancer compared with
GRPR-nonexpressing women with lung cancer was statistically significant (P =
.677).
As a control to determine if sex-specific expression of GRPR was related to its location on
the X chromosome, we also examined expression of the gene encoding the neuromedin B
receptor, another G-protein-linked receptor in the family of bombesin-like peptide receptors,
which is located on an autosome (chromosome 6). We were able to perform this analysis on
reverse-transcribed reaction products stored from 37 male subjects and 28 female subjects used in
the GRPR analysis (83.3% of all subjects). The stored RT reaction products were analyzed
for neuromedin B receptor mRNA in parallel with a new analysis for GAPDH, and all samples
used for neuromedin B receptor analysis showed a 520-bp GAPDH product (not shown). For five
subjects in the GRPR study, no further material was available; for eight subjects, the RT reaction
had deteriorated and was negative for GAPDH. The neuromedin B receptor results showed no
relationship to smoking, as we reported earlier (3): Nine (60%) of
15 lifetime nonsmokers were positive for neuromedin B receptor mRNA compared with four
(100%) of four short-term smokers (25 pack-years) and 21 (45.7%) of 46
long-term smokers. We also observed no significant sex difference for neuromedin B receptor
mRNA expression: Analyzed by gender, 80% (four of five) of male nonsmokers showed
neuromedin B receptor mRNA expression compared with 50% (five of 10) of nonsmoking
females. In short-term smokers, three of three males and one of one female were positive for
neuromedin B receptor expression. For long-term smokers, the frequency was 55.2% (16
of 29) in males and 29.4% (five of 17) in females. Although there was a trend for frequency
of neuromedin B receptor expression to decline after long-term smoking and to be lower in
females than in males, Fisher's exact test showed no statistically significant difference
between any of these groups. This result supports the hypothesis that the relationship between
smoking and GRPR expression frequency that we observed is specific to the GRPR locus and that
the gender differences may relate to the location of the GRPR gene on the X chromosome.
Nicotine, an important component of tobacco smoke, has been shown to directly induce expression of the genes encoding proenkephalin, diazepam binding inhibitor, and neutrophil elastase in cells that express nicotinic acetylcholine receptors, the sites through which nicotine exerts its biologic effects (29-31). It has also been shown that exposure to nicotine results in increased expression of a reporter gene under the control of the GRP gene promoter in U937 cells (36). It has recently been reported that human bronchial epithelial cells express nicotinic acetylcholine receptors (37). Because of the association that we observed between smoking history and GRPR mRNA expression, we hypothesized that nicotine might induce GRPR expression in the human airway and that human lung fibroblasts, like human bronchial epithelial cells, might express nicotinic acetylcholine receptors.
We examined binding of tritiated nicotine to human lung fibroblasts from one donor, human
bronchial epithelial cells cultured from six donors, and the immortalized human bronchial
epithelial cell line IB3-1. Consistent with a previous report (37), we found
saturable binding sites for nicotine in all bronchial epithelial cell cultures (data not shown). In
bronchial epithelial cell cultures, the number of specific binding sites per cell ranged from 5.4
x 104 to 15.1 x 104. Saturable, specific binding for
nicotine was also found in cultured lung fibroblasts (Fig. 2). The Kd (i.e., dissociation constant) for binding was approximately 30 nM. The
number of saturable binding sites was estimated at 1.3 x 104 per cell in lung
fibroblasts. We further determined the specificity of these nicotinic-binding sites by examining
competition for binding with nicotinic antagonists (Fig. 3
). At either 0.1 nM or 1.0 nM, tetraethylammonium chloride, (+)-tubocurarine, and
hexamethonium all displaced [N-methyl-3H]nicotine
binding to a statistically significant greater extent than unlabeled nicotine (P = .03
for tetraethylammonium chloride, P = .001 for (+)-tubocurarine, and P
= .01 for hexamethoniumtwo-tailed Student's t test), as would be
expected for authentic nicotinic acetylcholine receptors. These results suggest that human lung
fibroblasts, as well as human bronchial epithelial cells, express nicotinic acetylcholine receptors
and that, through these receptors, nicotine contained in cigarette smoke can have direct biologic
actions in the airway. A recent report (38) has noted that nicotine
stimulates branching morphogenesis in embryonic mouse lung cultures. Since GRP is known to be
crucial to branching morphogenesis in the developing lung, it is possible that the effects of
nicotine are mediated through induction of GRP and/or its receptor (39).
To determine if nicotine can alter expression of GRPR mRNA in airway cells, we examined
the effect of nicotine exposure on GRPR expression in cultured lung fibroblasts. Exposure of
cultured human lung fibroblasts to nicotine at levels found in the bloodstream of smokers (0.1
µM or 1.0 µM) resulted in an approximately 12-fold increase in
GRPR mRNA expression compared with that in control subjects [(29-31); Fig. 4]. These results suggest that nicotine in
cigarette smoke may be an important modulator of GRPR gene expression in the human airway.
We hypothesized that the second X-linked copy of the GRPR gene in women, if it is
functional, might be a target for induction by nicotine or other transcriptional regulators. To test
whether the increased frequency of GRPR mRNA expression in women is related to the
gene's location on the X chromosome and the fact that this locus escapes X-chromosome
inactivation in women, we examined a polymorphism involving two nucleotides in exon 2 of the
GRPR gene (26). We used allele-specific oligonucleotide hybridization to
detect these polymorphisms in DNA and RNA isolated from cells cultured from normal lung. Our
analysis of DNA from 26 females and two males confirms that males are hemizygous and that
approximately 38% of women are heterozygous at position 450 (can be a C or T) and
position 661 (also C or T), as previously described [(26); data not
shown]. We identified one female subject (sample 780) who is heterozygous at position 450
but homozygous at position 661; this recombinant genotype was also reported previously
[Table 4; (26)]. For nine subjects
(eight females and one male), both DNA and RNA were available for allele-specific
oligonucleotide hybridization analysis (Table 4
). For four heterozygous
women, GRPR mRNA was detected from both alleles, indicating that, in these women, the GRPR
gene copy on the inactive X chromosome might be expressed in the lung (Table 4
).
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DISCUSSION |
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The risk for all major lung cancer types is consistently higher for women than for men at every level of exposure to cigarette smoke and appears to be caused by an innate increased sensitivity to the carcinogenic effects of tobacco smoke (17). The airways of females appear more susceptible to the adverse effects of cigarette smoke than those of males (17,21). A study of lung function in adolescent smokers (40) suggested that, although smoking slowed the growth of lung function in both girls and boys, the deficits are greater in girls. The sensitivity of female lungs to tobacco carcinogens may be responsible for the observation that smoking cessation or the amount of lifetime smoking exposure affects the distribution of specific histologic subtypes of lung cancer to a greater degree in women than in men (41). Women are also at increased risk for other smoking-related malignancies, including esophageal and gastric cardia adenocarcinomas and esophageal squamous cell carcinoma (42,43). The risk for oral cancer following 40 or more pack-years of smoking was increased twofold in men and fivefold in women compared with nonsmokers (44). In addition to an increased risk for women due to smoking-related factors, there is evidence for an inherited genetic predisposition (45). Wang et al. (46) observed that a family history of cancer significantly increases the risk of lung cancer in nonsmoking Chinese women. Familial clustering of lung cancer has been observed among females and among nonsmokers, suggesting the involvement of a genetic component (47-49). It is possible that GRPR expression is determined by both genetic and environmental factors that could be related to the elevated risk observed among women and in some families or populations.
A number of studies have identified gender differences at the cellular level. Higher levels of aromatic/hydrophobic DNA adducts were observed in female lung cancer patients compared with males, even though the level of exposure to tobacco carcinogens was lower among the females than among the males (50). A higher frequency of G-to-T transversion mutations in the p53 gene has been observed in females compared with males (50). Gender differences have also been identified in the expression of the CYP1A1 gene, with females exhibiting higher mRNA levels in normal lung tissue than males, although large individual differences in expression level were observed (51). Immunohistochemical staining of c-erbB-2 was identified more frequently in female lung cancer patients than in male lung cancer patients (52). While these observations suggest that there are important molecular differences between men and women that may be related to lung cancer risk, no mechanisms have yet been shown to have a direct role in the etiology of lung cancer.
Although much work has been done to characterize the role of the GRPR-ligand system in cell growth, important gaps remain in our understanding of this system. There is strong evidence to support the hypothesis that the GRPR-ligand system plays a role in lung carcinogenesis, yet no studies have directly addressed the mechanism by which this pathway is activated and remains active after tobacco exposure ceases (3). Our results suggest that the second expressed copy of the GRPR gene in females may be involved in the increased susceptibility of women to tobacco-induced lung cancer. The ability to express the GRPR gene in the adult airway, either de novo or as a result of tobacco exposure, may be an underlying predisposing factor for development of lung cancer. The lack of either a smoking or a gender effect in expression of the neuromedin B receptor gene suggests that the observed effects on GRPR are specific and related to the escape of that gene from X inactivation and its ability to be induced by nicotine.
The increased frequency of GRPR mRNA expression in noncancerous airway cells and tissues from smokers with and without lung cancer suggests that GRPR expression may be an early event in the airway remodeling that takes place prior to development of lung cancer. Although it is still unclear whether GRPR expression alone is an important risk factor in healthy nonsmokers, GRPR mRNA expression may be a useful marker for increased lung cancer risk or preneoplastic change (either separate from or a part of histologic change). We have shown that the GRPR gene is expressed in PBLs and that the expression pattern in PBLs, which are also exposed to nicotine from cigarette smoke, may reflect that of the lung. If blood can be demonstrated to be a good surrogate for airway cells, large-scale screening of GRPR expression in at-risk populations would be possible.
We did not attempt to quantify the extent of mRNA expression in the airway cells from subjects in this study. This is because of the inherent difficulties in quantitating reactions relying upon amplification of cDNA and detection by Southern blotting. However, we did observe a subset of 12 subjects who yielded weak GRPR RT-PCR products. The intensity of these weak GRPR products (determined by densitometry) was less than 10% of the intensity of GRPR products from the majority of positive subjects, whereas the intensity of GAPDH RT-PCR products was approximately equal. Among these 12 subjects, seven were male and five were female. The group included two female subjects who were nonsmokers and one male subject who was a short-term smoker, as well as six subjects with smoking histories of 50 pack-years or fewer. The mean smoking exposure for this group was 48 pack-years for males and 38 pack-years for females. This observation suggests that there may be differences in amount of GRPR mRNA produced that are related to tobacco exposure, which could be detected with the use of a quantitative PCR technique with an internal standard. Such a technique, along with more complete exposure information about the subjects, is needed to determine more fully how GRPR expression is related to lung cancer risk.
Recent studies (53,54) in which morphology-based methods were used to determine the frequency of low- and high-grade lesions in the lungs of male and female current and former smokers have been inconclusive. Possible problems in relying only on morphologic alterations to screen for preneoplastic change include differences in pathologic interpretation and lack of objective criteria in interpreting fluorescence images. The inability to examine the peripheral lung, which is the usual site of adenocarcinoma development, may be critical in screening women for lung cancer, since lung cancer in women most often presents as adenocarcinoma (53). As a complement to morphologic criteria, molecular markers, such as GRPR expression, may prove to be useful in the detection of airway cells predisposed to lung cancer and may help to identify individuals at risk for developing lung cancer, especially women.
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NOTES |
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We are grateful to Ms. Camilla Chaparro for performing the neuromedin B receptor reverse transcription-polymerase chain reaction analysis.
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Manuscript received June 2, 1999; revised October 5, 1999; accepted October 28, 1999.
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