INTRODUCTION

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Bombesin-like peptides (BLPs) have been implicated in the growth of development of the fetal lung (1). GRP and NMB are two members of the BLP family that are found in mammals. GRP has been well documented as an autocrine growth factor for small cell carcinoma of the lung (5), and there is evidence that GRP is also a growth regulator of the adult human bronchial epithelium (8, 9). Our laboratory has also demonstrated that secretion of GRP occurs in NSCLC cells adapted to grow in growth factor-deprived medium (10). We have further documented the expression of mRNA for the three BLP receptors (GRP receptor, NMB receptor and bombesin receptor subtype 3 [11-13]) in human bronchial epithelium and in NSCLC cell lines (14). Recently, Wang and associates (15) have demonstrated that GRP receptor mRNA expression in the fetal rabbit and human lung was highest during times of airway growth and differentiation, and was localized to cells that were undergoing proliferation.

Several laboratories have demonstrated the presence of NMB immunoreactivity in NSCLCs (16, 17), as well as receptors for BLPs (18). Increased production of BLPs has been reported in the urine of asymptomatic cigarette smokers compared with nonsmokers (21). In addition, increased numbers of pulmonary neuroendocrine cells, positive for BLPs, have been reported in the airways of long-term smokers with bronchitis and emphysema compared to smokers or nonsmokers with no lung disease (22). These findings suggest that BLPs could play a role in the promotion of carcinogenesis in the adult lung. Recently, it has been reported that elevated expression of the GRP receptor following gene transfer to immortalized bronchial epithelial cells confers an increased growth response to GRP (23). Thus, increased expression of BLPs and their receptors could cause increased cell proliferation during the development of lung cancer. In this report, we demonstrate the association of prolonged tobacco exposure with expression of mRNA for the GRP receptor, and we demonstrate that the ability of both NMB and GRP to increase cell proliferation in HBE cells is associated with GRP receptor mRNA expression. These findings support the hypothesis that the GRP receptor-ligand system may contribute to lung carcinogenesis.

	METHODS

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Human Subjects Used for Study of Bronchial Epithelial Cells

Experiments were conducted using HBE cells cultured from 37 individuals (Table 1). These included 26 who underwent lung resection or bronchoscopy, eight who underwent lung transplantation, and three normal lung donors whose tissue was not used for transplantation. Of the 26 who underwent lung resection or bronchoscopy, 22 were diagnosed with lung carcinoma, two with a benign tumor of the lung (a thymoma and a leiomyoma), and two with carcinoma from a distant organ that metastasized to the lung. Of the eight lung transplant donors, three suffered from -1 anti-trypsin deficiency, one had scleroderma, two had chronic obstructive pulmonary disease, one had rheumatoid lung disease, and one had pulmonary hypertension. Tissues were collected under an approved institutional review board protocol. In cases involving lung resection or bronchoscopy, four bronchial biopsies were obtained from the major airways and placed into culture as described previously (24, 25). For individuals undergoing lung transplantation and tissue received from lung donors, the major bronchi from the removed lungs were dissected and scraped with a scalpel to release the bronchial epithelium. The bronchial cells were then cultured as for resection biopsies. Cultures were screened for mycoplasma containing and found to be negative.

Up to twenty 25 cm² flasks of primary HBE cells were obtained from each subject. These were then used simultaneously in up to three different assays, depending on the cell yield: a proliferation assay measuring colony-forming units, a "surrogate" assay of proliferative response measuring induction of the early response gene c-jun, and an assay of BLP receptor mRNA expression using RT-PCR. In three cases, all three assays were successfully performed. In five additional cases, the colony assay and the RT-PCR assay were performed. In two cases, the c-jun assay and the RT-PCR assay were performed. In 16 cases, only the colony forming assay was performed, and in 12 cases, only the RT-PCR assay was performed (see Table 1).

Medical histories were reviewed to obtain information on smoking history, smoking status at the time of tissue procurement, diagnosis, existence of other disease, and pulmonary function. Pulmonary function tests were reviewed by a pulmonologist (J.M.P.) and the subjects were categorized according to normal function (80% or greater of predicted FEV₁, and FEV₁/FVC ratio > 0.7), moderate obstruction (60-79% of predicted FEV₁, and FEV₁/FVC ratio of < 0.7), severe obstructive disease (< 60% of predicted FEV₁, and FEV₁/FVC ratio < 0.6), or restrictive lung disease (FEV₁ and FVC < 80% of predicted, and lung volumes < 80% of predicted). For the three normal donor lungs that were not used for transplant, limited information was available.

Proliferation Assays

Colony-forming assays were performed as described (24, 25). Briefly, single-cell suspensions of HBE cells obtained from primary cultures were plated in 35 mm culture dishes (1,000 cells/well) using basal medium as described (24, 25). Dishes contained a confluent layer of inactivated Swiss 3T3 cells (24). Each condition was measured in triplicate. GRP or NMB diluted in saline was added to wells at the indicated concentration; in the control, saline alone was used. Cultures were allowed to form colonies over a 10-d period, at which time the cells were washed with saline, fixed in methanol, and stained with Giemsa as described (24, 25). Colonies were enumerated under a dissecting microscope. If a statistically significant increase in colony formation was observed (p  0.05), the culture was considered to have given a positive response. Otherwise, it was considered to be nonresponsive.

In some cases, induction of the early response gene c-jun was used to assess a positive response to BLPs as described previously (14). We (14) and others (26) have shown that BLPs induce c-jun expression in lung tumor cells that proliferate in response to BLPs. Briefly, subconfluent cultures, of HBE cells were placed in basal medium as described (14) for 16 hr. Different concentrations of BLPs, other growth factors as positive controls, or saline as a negative control were added to duplicate flasks of the deprived cells and incubated for 30 min at 37° C. This timing has been found to be optimal for c-jun induction (14). Cells were washed with saline, RNA was isolated as described (14), and northern analysis was performed as described (14) using the plasmid pHj, containing the cDNA sequence for c-jun. The signal from the hybridization with the pHj plasmid was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. If the increase in c-jun signal in response to BLP was at least 1.5-fold higher than the control signal after normalization for the housekeeping gene GAPDH, the culture was considered to have given a positive response. Otherwise, it was considered to be nonresponsive.

BLP Receptor mRNA Expression

Expression of mRNA for the three BLP receptor subtypes was analyzed as described (14) using RT-PCR. Because these assays were performed with primary HBE cells, it was not possible to measure protein levels of the receptors. To differentiate among the receptor subtypes, which cannot be done at present with binding assays or antibodies, we used the sensitive RT-PCR technique under conditions that can specifically detect the three receptor subtypes. The primers and conditions were reported previously (14). Briefly, total RNA was used to produce single-stranded cDNA by reverse-transcription, and H₂O was used as a negative control. The cDNA was then amplified by PCR using Amplitaq DNA polymerase. The PCR conditions were: 30 cycles total, comprised of denaturation at 94° C for 1 min, annealing at 55° C for 2 min, and extension at 72° C for 2 min in a thermocycler. Southern analysis was performed with the appropriate cDNAs (14) to examine the products and determine their sizes. The PCR products were subjected to get electrophoresis in a 3% NuSieve 3:1 Tris-boric acid EDTA gel, transferred to Nytran filters, and hybridized to the appropriate probe as described (14). For the GRP and NMB receptor mRNAs, either H345 cells or IB31 cells (immortalized human bronchial epithelial cells) were used as a positive control, and for the BRS3 receptor mRNA, IB31 cells were used as a positive control (14). If a band was visible of the appropriate size after hybridization, the sample was considered to be expressing message for that receptor. As a control for the integrity of the RNA and the success of the reverse transcription step, the GAPDH gene was also amplified from RNA. In all cases, the RNA for GAPDH was successfully amplified.

	RESULTS

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BLP Receptor mRNA Expression

To detect expression of mRNA for the three BLP receptor subtypes, RT-PCR was performed using HBE cells cultured from 22 subjects (Table 1). For the GRP receptor, the PCR product from one subject was lost and not analyzed. Of the cultures examined, 12 were positive for GRP receptor mRNA, 12 for NMB receptor mRNA, and five for BRS3 receptor mRNA (Table 1). Examples of RT-PCR products for each receptor subtype are shown in Figure 1, with positive and negative cultures. In these examples, cells from Patient 1, Patient 3, Patient 6, and Patient 7 are positive for the GRP receptor mRNA, showing the correct band size at 390 bp, while other cultures are negative (Figure 1A) (Table 4). For the NMB receptor (Figure 1B), IB31 cells and cells from Patient 1 are positive for mRNA in this example, showing the 424 bp PCR product, while cultures from the other five subjects are negative. None of the cultures examined in this example were positive for the BRS3 receptor mRNA except the control IB31 cells and the small cell lung cancer cell line H345 (Figure 1C). Smaller molecular weight bands seen in the figures represent single-stranded PCR products, which migrate differently than double-stranded products (14).

View larger version (45K): [in this window] [in a new window]   View larger version (58K): [in this window] [in a new window]   View larger version (62K): [in this window] [in a new window]   Figure 1.   Expression of BLP receptor subtypes analyzed by RT-PCR. Conditions were as described in METHODS. (A) Expression of the GRP receptor mRNA in HBE cultures from six patients (390 bp RT-PCR product); (B) Expression of the NMB receptor mRNA in HBE cultures from six patients (424 bp RT-PCR product); (C  ) Expression of the BRS3 receptor mRNA in HBE cultures from five patients (489 bp RT-PCR product).

Expression of mRNA for the receptor subtypes was analyzed in relation to smoking history (Table 2). There was an increased frequency of expression of mRNA for the GRP receptor in cultures from subjects with lengthy smoking exposure. Only one of four nonsmokers expressed the GRP receptor mRNA. None of three cultures from subjects with smoking histories of 25 PY or less expressed the GRP receptor mRNA, whereas 10 of 13 cultures from subjects with more than a 25 PY tobacco exposure were positive. Comparing those with more than 25 PY tobacco exposure with those with a 25 PY history or less, there was a significant difference in GRP mRNA expression (p = 0.02). A comparison of all smokers versus nonsmokers was not significant (p = 0.13). This implies that duration of smoking is an important variable in GRP mRNA expression, not smoking alone. The expression of NMB and BRS3 receptor message was not related to tobacco exposure (p = 0.67 and p = 0.88).

Smoking cessation also did not appear to affect the expression of the GRP receptor message (Table 2). Of cultures from the 16 subjects with positive smoking histories that were used for RT-PCR analysis of GRP mRNA, 11 had undergone smoking cessation for 2 yr or more, including three subjects with over 20 yr of smoking cessation. Of the 13 subjects used for mRNA expression assays whose smoking histories were greater than 25 PY, nine were ex-smokers for at least 2 yr. Comparing the pattern of expression of the ex-smokers with active smokers and nonsmokers, ex-smokers continued to express the GRP receptor mRNA. When ex-smokers were combined with nonsmokers for statistical analysis, there was no significant difference in the pattern of GRP receptor mRNA expression between active smokers and nonsmokers, indicating that the increased incidence of GRP receptor mRNA expression observed in subjects with lengthy smoking histories is still present after tobacco exposure ceases.

Assays of Proliferative Response of BLPs

In colony assays using HBE cells, a positive, statistically significant response to BLPs was found in cultures from 10 individuals of 23 tested (Table 1). Cultures from thirteen individuals gave either no response or a marginally positive response that was not significant at the 95% confidence level. In assays of induction of c-jun, three of five individuals showed a positive response (Table 1). This assay was performed less frequently because of the number of primary cells required. Cultures that responded to NMB also responded to GRP, usually with the same magnitude. Examples of colony assays using HBE cells cultured from three different responding individuals are found in Figure 2. Statistically significant increases in colony forming efficiency (the number of colonies formed per well) were found using both NMB and GRP. The maximum response was a 2.9-fold increase using HBE cells from Patient 1, a lung cancer patient (10 nM GRP and 10 nM NMB, Figure 1A), a 3.6-fold increase using cells from Patient 2, another lung cancer patient (1 nM GRP and 1 nM NMB, Figure 1B) and a 1.9-fold increase using cells from Patient 3, a lung transplant patient with emphysema (5 nM GRP and 20 nM NMB, Figure 1C). The level of significance was p < 0.01 in Patient 1, p < 0.05 in Patient 2, and p < 0.01 in Patient 3. 

View larger version (65K): [in this window] [in a new window]   View larger version (100K): [in this window] [in a new window]   View larger version (76K): [in this window] [in a new window]   Figure 2.   Positive response of HBE cells from three patients to BLPs in the colony-forming assay. (A) Patient 1; *p < 0.01; (B) Patient 2; *p < 0.05; (C  ) Patient 3; *p < 0.01. Data were analyzed by Tukey-Kramer multiple comparison test.

Examples of a positive response and a nonresponse in the c-jun induction assay are found in Figure 3. In panel A, cells from Patient 5, a patient with a benign thymoma, were used. A 5-fold induction of c-jun signal was observed with GRP. This was greater than that observed with two control growth factors, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I). In panel B, cells from Patient 9, a lung transplant patient with rheumatoid lung disease, were used. In this case, although EGF elicited an increase in c-jun signal, NMB did not, either alone or in combination with EGF. Good agreement was seen between a positive response to BLPs in the colony assay and the c-jun induction assay in the three cases where both assays were performed.

View larger version (42K): [in this window] [in a new window]   View larger version (59K): [in this window] [in a new window]   Figure 3.   Response of two HBE cultures in the c-jun induction assay. (A) Positive response of Patient 5 to GRP and NMB; (B) Negative response of Patient 9 to NMB.

When smoking history was examined, there was an increased likelihood of a positive response to BLPs in cells from subjects with a lengthy exposure to tobacco (Table 3). A positive response was seen in one of the four cultures from a lifetime nonsmoker, in none of the three smokers with 25 PY or less smoking history, and in 10 to 16 smokers with more than a 25 PY smoking history. Comparing the number of positive responses in cultures from subjects with greater than a 25 PY history with the number of positive responses in cultures from subjects with a 25 PY or less exposure (10 of 16 versus one of seven), a strong trend was found that was not quite statistically significant (p = 0.06, Fisher's exact test). The small number of nonsmokers whose cells were available for this assay was a factor in the lack of statistical significance. Removing non-lung cancer patients or those with -1 anti-trypsin deficiency, who may not have the same risk factors for lung cancer than the other subjects, did not improve the statistics.

We also examined whether smoking cessation was a factor in a positive response to BLPs (Table 3). There were 10 ex-smokers in the smoking group whose period of smoking cessation was at least 2 yr, including seven whose previous smoking history was more than 25 PY. Of these individuals, five showed a positive response to BLPs (50%, Table 3), including two with smoking cessation periods of 10 and 25 yr, while of the 10 active smokers, or those with smoking cessation periods of less than 2 yr, six showed a positive response (60%, Table 3). This implies that the trend toward heightened responsiveness of individuals with lengthy smoking histories persists after smoking cessation, in agreement with the results of GRP mRNA expression.

For 10 individuals from whom bronchial tissue was procured, data were obtained both for proliferation responses and for the pattern of BLP receptor subtype mRNA expression (Table 4). Cells from six of the subjects gave a positive response in either the colony assay, the c-jun induction assay, or both, while cells from four of the subjects were negative in these assays. (Table 4). Of the six cultures with a positive proliferative response, five expressed GRP receptor mRNA, while none of the four nonresponsive cultures were positive for GRP receptor mRNA. The association of GRP receptor mRNA with a positive proliferative response was significant (p = 0.048). Four of six of the responsive cultures also expressed the NMB receptor message, compared to two of four in the nonresponsive group. This difference was not significant. The BRS3 receptor mRNA was only expressed by one nonresponsive culture. Of the six responsive cultures, three expressed both the GRP and NMB receptor mRNA, while none of the nonresponsive cultures expressed both receptors, but this difference was not significant. Two nonresponsive cultures expressed no mRNA for any of the BLP receptor subtypes. All six of the responsive cultures showed a proliferative response to both GRP and NMB, even if mRNA for only one of these receptor subtypes was expressed.

Lung Function as a Factor in Responsiveness to BLPs and Expression of BLP Receptor mRNA

Records of pulmonary function tests were found for 32 patients (Table 5). Cells from subjects who had severe obstructive lung disease associated with smoking showed a greater tendency to respond to BLPs (four of five positive responses) compared with cells from other subjects with normal lung function (four of nine), moderate obstruction (one of three), and reduced lung function due to restriction of the airways not associated with smoking (one of four), but this trend was not significant (p = 0.13). If individuals with -1 anti-trypsin deficiency were excluded, the statistical association between obstruction and BLP responsiveness was not improved, suggesting this group is not masking an effect seen in individuals whose airway obstruction is more directly related to lung cancer risk. There were no differences in expression of mRNA for the three bombesin receptor subtypes among subjects with varying pulmonary function (Table 5). In particular, expression of mRNA for the GRP receptor was not confined to those with obstructive lung disease. This result suggests that the increased incidence of GRP receptor mRNA expression does not occur as a result of the development of obstructive disease.

We also examined a possible association between the presence of lung cancer and expression of mRNA for BLP receptor subtypes, as well as responsiveness to BLPs. Comparing individuals with and without lung cancer showed no statistical association with any of the receptor subtypes or with responsiveness to BLPs. This finding further supports the hypothesis that, at least in this group of patients who are undergoing resection or lung transplantation, duration of smoking is the most important variable in expression of the GRP receptor mRNA and responsiveness to BLPs.

	DISCUSSION

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BLPs have been implicated in the growth of the fetal lung (1- 4, 15, 27). Administration of BLPs to explants of human and murine fetal lungs resulted in increased growth and maturation of the airways (4). Branching morphogenesis also increases in explants of fetal mouse lung cultured with BLPs (27). In addition to effects on lung development, BLPs may be important regulators of lung tumor growth. The proliferative effects of BLPs on SCLC cells is well documented (5), and evidence is accumulating that BLPs are also involved in the development of NSCLC (28, 29). Despite having different clinical manifestations, SCLC and NSCLC share many properties and may originate from a common stem cell (28, 30). Experimental manipulation has shown that SCLC can be induced to display a NSCLC phenotype by introduction of a mutated H-ras gene (30), suggesting there is plasticity in the phenotypes that lung tumors can express. Our laboratory has recently demonstrated that NSCLC that normally do not produce GRP can be induced to actively secrete processed GRP when cultured in medium devoid of all growth factors (10). Again, these observations support the concept of plasticity within the lung epithelium and common mechanisms of carcinogenicity for all types of lung tumors (31).

One common etiological factor for different types of lung cancer may be the proliferation of cells that produce and/or respond to BLPs. Urinary levels of BLPs are increased in smokers compared with nonsmokers (21, 32). In disease states associated with smoking, neuroendocrine cells that produce BLPs are increased (22, 33). For example, individuals with chronic bronchitis and emphysema showed increased numbers of pulmonary neuroendocrine cells containing GRP and calcitonin (22). Cells of monocyte and macrophage lineage also show increased BLP content in individuals with chronic bronchitis compared to normal controls (36). Thus, in diseases of the lung, increased BLPs may come from the lung itself or from inflammatory cells infiltrating the lung.

The results of our study demonstrate that the BLP peptides GRP and NMB are both mitogens for normal adult bronchial epithelial cells cultured from some, but not all, individuals, and that an important determinant is expression of the GRP receptor, as opposed to the two other BLP receptor subtypes. In this group of 37 bronchial tissue donors, expression of the GRP mRNA is more common in chronic smokers with a greater than 25 PY tobacco exposure than in those with a smoking history of 25 PY or less (including nonsmokers). Our data suggest that expression of the GRP receptor is related to the ability of BLPs to induce proliferative responses in HBE cells, which were also more common in individuals with lengthy smoking histories. While induction of GRP receptor expression could be a response to the documented increase in production of BLPs in chronic smokers (21), it is also possible that the pattern of GRP expression observed in this study preexisted in the individuals studied, and that a bias toward lengthy smoking history was introduced due to the fact that these individuals had airway disease and/or lung cancer which caused them to seek surgical treatment and become subjects in this study. Whether the same pattern would be observed in the general population of smokers is not known.

In contrast to our results that a proliferative response to GRP and NMB could be demonstrated in HBE cultures from almost half the bronchial tissue donors in our study, Frankel and associates (37) reported no effect of bombesin on intracellular calcium concentration or proliferation in HBE cultures from six individuals. The smoking history of the individual donors might have been a factor in their results, or possibly growth of cells in mass culture is not sensitive enough to pick up responses to BLPs.

Our results also suggest that although GRP receptor expression is associated with prolonged smoking, the expression of the NMB and BRS3 receptor is not. Although little is known about the importance of the NMB and BRS3 receptors during lung development, there is considerable evidence that the GRP receptor is expressed at crucial times during lung budding and formation of the airways (15, 27), and that it is expressed by the cells that are undergoing proliferation (15). Our results give further support for a role of the GRP receptor in proliferation of the adult bronchial epithelium, and suggest that the GRP ligand-receptor system is an important factor in changes to the airways induced by smoking.

Smokers with reduced lung function have an increased risk of lung cancer compared to smokers with normal lung function, and BLPs may play a role in this susceptibility (34). We analyzed lung function of tissue donors as a factor in responsiveness of cultured HBE cells to BLPs and expression of mRNA for BLP receptors. We found that obstruction of the airways caused by tobacco exposure was only marginally associated with responsiveness to BLPs, and no association of pulmonary function with expression of the GRP receptor was found. This suggests that the effects we observed in chronic smokers were not indirectly due to underlying airway disease, and it is possible that biological response to BLPs is one of the factors that contributes to the ultimate development of airway obstruction. Although we had only a limited number of donors with lung pathology not associated with smoking, we did not see differences in the incidence of response to BLPs and expression of the GRP receptor in these individuals compared to the normal lung donors. This implies that the effects we observed in long-term smokers are not associated with lung pathology in general.

Our results also suggest that in the long-term smokers who showed an increased GRP receptor expression and proliferative responsiveness to BLPs, the effects persist after smoking cessation. The percentage of ex-smokers who showed a positive response to BLPs in the proliferative assay and who expressed mRNA for the GRP receptor was the same as the active smoking group. Of the 10 individuals with a greater than 25 PY history whose HBE cells expressed the GRP receptor mRNA, seven were ex-smokers, including a subject who was an ex-smoker for 25 yr. This could be explained either by an induction mechanism that remains activated, or by a genetic predisposition that is permanent. Regardless of the mechanism, an enhanced responsiveness to BLPs could persist as a risk for cancer in causing continual proliferative changes in the airways of smokers.