Receptor Subtype Expression and Responsiveness to Bombesin in Cultured Human Bronchial Epithelial Cells1

(CANCER RESEARCH 54. 1613-1616. April 1. 1 Advances in Brief

Andrea Franke!, Ming-Sound Tsao, and Jean Mailer Dffwrtmcnts of Oni'ologv fj. VJ, Medicine Â¡A.F., J. VJ, ami Pathology fM-S. T.j, Montreal General Hospital and Research Institute and McfÃ¬illuniversity, MontrÃ©al.P.Q., Quebec, H3C 1A4 Canada

Abstract Materials and Methods

We detected low level expression of the gastrin-releasing peptide and Materials. KSF medium with its supplements of BPE and human recom neuromedin-B receptor mRNAs in cultures of human bronchial epithe binant EGF were purchased from G1BCO-BRL (Grand Island, NY). lium from 4 of 6 individuals. Bombesin receptor subtype-3 mRNA was [Tyr4]Bombesin and neurotensin were from Bachern. Inc.(Torrance. CA). undetectable in these cells. An elevation of intracellular calcium concen Vasopressin, bradykinin, cholecystokinin, and Quin-2 AM were from Sigma (St Louis. Mo). CT was from List Biologicals, Inc. (Campbell CA). [32P]dCTP tration was observed in response to bradykinin (6 of 6) and neurotensin (1 and Hybond-N membranes were from Amersham Canada (Oakville, Ontario. of 5) but not to bombesin (0 of 6), vasopressin (0 of 6), or cholecystokinin (0 of 3). In contrast, such responses are frequently noted in lung cancer Canada). Alamar Blue was from BioSource International (Camarillo, CA). cell lines. Bombesin did not stimulate the in vitro growth of an immortal Cell Culture. The primary cultures of HBE cells were established as described previously (10) and maintained in KSF medium supplemented with ized human bronchial epithelium cell line expressing low levels of bomb EGF (5 ng/ml), BPE (50 /xg/ml), and CT (10 ng/ml). Neither precoating of esin receptor mRNAs. We conclude that bombesin receptors are expressed plates with extracellular protein matrix preparation nor antibiotics were used. at low levels in human bronchial epithelium cells which may acquire greater responsiveness to multiple neuropeptides in the course of multi- We also used a single, immortalized HBE cell line, which we established by transfection of a pl321 plasmid construct expressing (he human papillomavi- step carcinogenesis. rus type 16 Â£6and Â£7genes (11, 12). Primary and transfected cells were grown under identical conditions and tested negative in a test for Mycoplamna Introduction contamination (Mycotec: GIBCO-BRL, Grand Island, NY). SCLC cell lines NCI-H345 and N417 were obtained from the American Type Culture Collec The involvement of bombesin-like peptides in lung cancer biology tion and maintained in RPMI 1640 (GIBCO-BRL, Grand Island, NY) supple has been extensively studied for the past decade (1). These peptides mented with selenium, insulin, and transferrin (selenium insulin transferrin) (gastrin-releasing peptide and neuromedin-B) may be produced by (4). All cell cultures were maintained at 37Â°Cin an atmosphere containing 5% lung cancer cells which often express one or more of the three known CO,. bombesin receptor subtypes, creating the opportunity for autocrine Reverse Transcription-PCR Analysis of mRNA Expression. Total RNA growth stimulation. The cloned receptors can be distinguished by their (2 jig) prepared from primary HBE cultures or cell lines was reverse tran relative affinity for specific ligands. They are classified as the GRP-R1 scribed using the first-strand complementary DNA synthesis kit from Phar- macia-LKB (Piscataway, NJ) according to the manufacturer's instructions. (2), the NMB-R (2), and the BRS-3 (3), the natural ligand of which is Subsequent steps of PCR amplification and Soulhern blotting were as de not yet known. All three subtypes belong to the serpentine receptor scribed previously (3). Oligonucleotides corresponding to sequences situated superfamily and are coupled through G-proteins to effectors including between those used for amplification served as probes. Primers were chosen on phospholipase C. A stimulation of lung cancer cells with bombesin either side of first intron sequences to ensure that only complementary DNA results in the activation of phospolipase C, enhanced turnover of could serve as a template for PCR. phosphoinositides, and transient elevation of the free [Ca"+]i (4, 5). For GRP-R, .V-ATCTTCTGTACAGTCAAGTCC-3' (nucleotides 190-210), Bombesin-like peptides may be involved in the earliest stages of 5'-GCTTTCTCATGGAAGGGATG-3' (nucleotides 543-564), and human bronchial carcinogenesis. They are found in excessive amounts 5'-GATGCCAGCAGGTACCTGGCT-3' (nucleolides 287-308) were used as a probe. in the bronchial secretions of smokers (6), perhaps secreted by hy- For NMB-R. S'-ATCACCAACAGCGCCATGAGG-S' (nucleotides 205-226), perplastic bronchial neuroendocrine cells, which often occur in dis 5'-CTTTGGATGTAATTCATCTGT-3' (nuclcotides 597-618). and eased airways (7). Bombesin has been reported to stimulate or inhibit S'-GAAGTGGCTCGCATCAGTAGC-S' (nucleotides 547-566) the clonogenic growth in vitro of HBE cells obtained from different donors (8, 9). We report that bombesin receptor subtypes are ex were used as a probe. For BRS-3, Oligonucleotides for amplification and pressed at very low levels or not at all in HBE cells. In addition, HBE hybridization were as described previously (3). cells do not possess a [Ca2<~]Â¡response to bombesin and other neu Determination of [Ca2*]Â¡Responses. HBE cells were grown in KSF medium supplemented with EGF (5 ng/ml), BPE (50 fig/ml), and CT (10 ropeptides as is frequently observed in lung cancer cells. ng/ml) until 24 h before the experiment when cells were washed and rel'ed with KSF medium with no added supplements. Cells were grown on coverslips and Received 11/15/93; accepted 2/21/94. loaded with Ouin-2 by incubation with 5 /MMof the indicator for 15 min before The cost of publication of the article were defrayed in part by the payment of page extensive washing. The coverslips were then introduced in a spcctrophotom- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. eter cuvette containing a buffer composed of 140 mM NaCI, 5 mM KC1. 5 mM 1Supported by Grant MT-11377 from the Medical Research Council of Canada and glucose, 1 mM CaCl2, 1 mM MgCl,, and 20 mM 4-(2-hydroxyethyl)-l-pipera- the Faculty of Graduate Studies, McGill University. J. V. is a Scholar of the Medical zineethanesulfonic acid at pH 7.4. Measurements were performed with a Research Council of Canada. ~ To whom requests for reprints should be addressed, at The Montreal General Perkin-Elmer LS50 Luminescence Spectrometer with an excitation wavelength Hospital, 1650 Cedar Avenue, Montreal, P.O., H3G 1A4 Canada. of 339 nM and an emission wavelength of 490 nM. Slit widths were set at 5.0 ' The abbreviations used are: GRP-R, gastrin-releasing peptide receptor; NMB-R, and 10.0 mm, respectively. In some experiments, cells were loaded with neuromcdin-B receptor; BRS-3, bombesin receptor subtype-3; [Ca:< ]Â¡,inlraccllular cal Ouin-2 under the same conditions, briefly incubated with phosphate-buffered cium concentration; HBE. human bronchial epithelium; KSF. keratinocyte serum-free; salinc-1 mM EDTA to produce a crude cell suspension, which was resuspendcd BPE, bovine pituitary extract; EGF, epidermal growth factor; CT, cholera toxin; SCLC. smalli cell lung cancer; PCR. polymerase chain reaction; NSCLC, non-small cell lung in experimental buffer and monitored in the same fashion. Results obtained cancer. with the two methods were identical and were pooled for presentation. 1613

1 23456789 cultures prevented, on occasion, a comprehensive study of cells from all donors. GRP-R -374 bp Fig. 2 shows a typical tracing of [Ca2*]Â¡after sequential stimulation with neuropeptides. In this particular example, HBE 1 cells (sixth expiant, passage four) showed a responsiveness to the addition of NMB-R -413 bp bradykinin. The cells were refractory to a second addition of brady kinin and exhibited homologous desensitization as reported previ ously for lung cancer cells (4, 13). These cells, however, show no response to other neuropeptides including bombesin, vasopressin, and BRS-3 -489 bp neurotensin. They are sensitive to serum. Fig. 1. Expression of bombesin receptor subtype mRNA in HBE cells. Lane 1, water; Lane 2, HBE-1; Lane 3, HBE-2; Lane 4, HBE-4; Lane 5, HBE-5; Lane 6, HBE-6; Lane Our results of repeated experiments with up to six different HBE 7, HBE4-E6/E7; Lane 8, NCI-H345; Lane V, NCI-N4I7. cell populations are presented in Table 1. All HBE cells responded to the exogenous addition of bradykinin. In contrast to lung cancer cell lines of both SCLC and NSCLC origin, which frequently respond to Mass Culture Assay for Cell Growth. Cell growth was assayed by plating HBE4-E6/E7 cells at a density of 4,000 cells/well into 96-well tissue culture a combination of bombesin, vasopressin, cholecystokinin, and neuro plates in KSF medium without added EOF, BPE, or CT. EOF or tensin (13, 14), only the immortalized HBE cell line responded to [Tyr4]bombesin was then added at the final concentrations indicated. After 5 neurotensin. We observed no [Ca2+]Â¡responses to any of the other days of incubation, cell population density was scored by adding the vital peptides in up to five HBE cell cultures and the single cell line colorimetrie indicator Alamar Blue to the wells. The resulting absorption was studied. These results show that the low level of bombesin receptor read with an enzyme-linked immunosorbent assay reader according to the subtype mRNAs observed in the HBE cells is not sufficient to elicit all manufacturers recommendation. The mean of eight replicate wells was calcu of the responses observed in lung cancer cells. There seems to be a lated for each experiment. recruitment of responsiveness to neuropeptides, at least in terms of elevations of [Ca2 +]Â¡,which occurs during transformation of HBE Results cells. Previous work had shown that the level of mRNA expression of the We then examined the effect of bombesin on the proliferation of bombesin receptor subtypes in lung cancer cells is very low, and their HBE cells. Because of the brief time span of HBE cells in primary detection requires RNase protection assay or the PCR amplification of culture, we limited our study to the immortalized cell line. Fig. 3 reverse transcribed total cellular mRNA (2, 3). In order to obtain the shows that the growth of these cells can be stimulated approximately greatest possible sensitivity, we chose the latter method to explore the 3-fold by the exogenous addition of EOF, demonstrating that this relative expression of the bombesin receptor subtypes in five HBE assay is sensitive enough to detect mitogenic stimulation by exoge primary cell cultures established from different individuals, an im nous growth factors. However, exogenous bombesin did not signifi mortalized HBE cell line established after transfection with human cantly stimulate the cell proliferation. This is consistent with the lack papillomavirus type 16 E6 and E7 genes, and two human SCLC cell of [Ca2+]Â¡responsiveness and the very low level of expression of lines previously shown to express the various subtypes. bombesin receptor subtype mRNA in this cell line (Fig. 1, Lane 7). Successful amplification of ÃŸactin sequences demonstrated the Finally, we could detect no specific binding of [125ITyr4]bombesin in efficacy of the reverse transcription reaction (data not shown). The this cell line (data not shown). same reverse transcription products were then used to study the expression of the bombesin receptor subtypes by PCR amplification, followed by Southern blotting and hybridization with 32P-labeled oligonucleotide probes. Fig. 1 shows the variable expression of the 0. < GRP-R and NMB-R subtypes, albeit always less than the SCLC > rr controls. Two of the six HBE samples did not express the GRP-R or the NMB-R subtypes. None of the HBE samples expressed the BRS-3 800 n subtype. Fig. 1 probably underestimates the differences of expression between HBE cells and NCI-H345. The appearance of Fig. 1, Lanes 300 - 8 and 9, would suggest comparable expression between NCI-H345 and N417, whereas previous work has shown that GRP-R and NMB-R 150 J messages in H345 were easily detected by RNase protection assay, 5* but they were undetectable in N417 (2). These experiments suggest that expression of the bombesin receptor subtypes is greater in lung Fig. 2. [Ca2 +]j responses to neuropeptides in HBE cells. A representative tracing obtained from HBE-1 cells. Scale on the left, [Ã‡a2"1"];innM. Additions were made in the cancer cells than in HBE cells. The total lack of expression found indicated sequence: [Tyr4]bombesin (BOM), vasopressin (AVP), bradykinin (BRA), neu for the BRS-3 subtype constitutes an additional evidence that the rotensin (NEU), serum (FCS), and ionomycin (IONO). aberrant expression of bombesin receptors is induced during bronchial carcinogenesis. Bombesin stimulation typically results in a transient elevation in Table 1 Prevalence of Â¡Ca2*Â¡Â¡responsesto neuropeptides in normal bronchial [Ca2+]j in lung cancer cells (4). In fact, lung cancer cells are charac epithelial cells terized by a broad [Ca2 +]Â¡responsiveness to multiple neuropeptides HBE" Peptide including bradykinin, vasopressin, neurotensin, and cholecystokinin Bombesin 0/6 (13, 14). In order to determine whether the low levels of bombesin Bradykinin 6/6 receptor subtype mRNA observed in HBE cells could result in the Vasopressin O/6 Cholecyslokinin 0/3 activation of intracellular signals after stimulation with bombesin, we monitored [Ca2"1"^responses in HBE cells after exposure to bombesin Neurotensin 1/5 " Includes five HBE primary cultures (HBE-1, -2, -4, -5, and -8) and one immortalized and other neuropeptides. The limited lifespan of primary HBE cell HBE cell line (HBE4-E6/E7). 1614

resections for lung cancer or pulmonary transplantations for severe chronic obstructive pulmonary disease. It is among the latter, whose bronchial epithelium showed marked pathological changes of glandu lar hyperplasia and squamous metaplasia, that the most significant growth stimulation by bombesin was noted. The only specimen ob c tu O tained from a normal lung donor showed an inhibition of clonal o . growth in response to bombesin. Although the samples we studied li were all obtained at the time of pulmonary resection for lung cancer, primary cultures were obtained from grossly normal mucosa, a sig nificant distance away from the tumor, and without significant patho logical changes. These observations support our interpretation that greater responsiveness to bombesin and other neuropeptides is ac quired during the progressive multistage carcinogenesis of human bronchial mucosa. In view of the poor understanding of the relation ships between the cellular components of the normal bronchial epi O/O 1/0.1 10/0.5 100/1 500/5 thelium and the various histologies of lung cancer, it would be [Bombesln] (nM) / [EGF] (ng/ml) interesting to study the relative expression of bombesin receptor subtypes in situ in bronchial epithelium exhibiting different preneo- Fig. 3. Effects of [Tyr4|bombesin and EGF on Ihc growth of HUE cells. HBE4-E6/E7 cells were exposed lo the indicated concentration of growth factor for 5 days. The density plastic changes. Particularly, the acquisition of expression of the of the cell population was then measured and is expressed relative to an untreated control BRS-3 receptor subtype might represent an intermediate marker of (mean Â±SI: of 3 independent experiments). Bars, SE. bronchial carcinogenesis. The precise contribution of overexpression of bombesin receptor Discussion subtypes to lung cancer biology can be addressed by transfection experiments with the cloned genes. Whether this will be sufficient or We have shown that, in mass cultures of HBE cells, bomhesin not to transform immortalized HBE cells is open to question since receptor subtypes are expressed at very low levels. In particular, the analogous experiments with the receptor tyrosine kinase c-erhB-2 BRS-3 subtype, which was previously demonstrated to have very proved to yield only poorly tumorigenic clones (15). Additional restricted expression in the rat testis, could not be detected in HBE disturbances in the signal transduction machinery may be necessary cells, despite its ubiquitous expression in lung cancer cell lines (3). before the full mitogenic potential of bombesin and other neuropep Furthermore, we have shown that the low levels of expression of these tides in epithelial cells may be realized. However, the transition from receptors in the HBE cells we studied are not sufficient to trigger an [Ca2^ ]Â¡response or growth stimulation after exposure to bombesin. a neuropeptide unresponsive state in HBE cells to a responsive state A previous report of the pattern of [Ca2 ' ]Â¡responses to neuropep- in lung cancer cells identifies a possible biological target of some specificity for the development of new approaches into the prevention tides in lung cancer cell lines helps to put our findings in perspective and treatment of lung cancer. (14). Responses to bradykinin were noted in 5 of 5 NSCLC and 8 of 13 SCLC cell lines, indicating that the sensitivity to bradykinin noted Acknowledgments in HBE cells is usually maintained in lung cancer cells. In contrast to the HBE cells we studied, NSCLC and SCLC cell lines were found to We thank Lyne Boucher and Dr. Michael Antccol for assistance in the conduct of these experiments and Dr. li. W. Bradley of the InstituÃdu C'ancer be frequently responsive to bombesin (2 of 5 and 8 of 13), vasopressin (2 of 5 and 7 of 13), cholecystokinin (2 of 5 and 9 of 13), and de MontrÃ©alfor having provided the oligonucleotides used in our studies. neurotensin (3 of 5 and 9 of 13). When compared to these previously published data, the results of Table 1 suggest that a greater respon References siveness to neuropeptides is a feature accompanying bronchial cell 1. Viallet. J.. and Minna, J. Gastrin-releasing peptide (GRP, mammalian bombesin) in the pathogenesis of lung cancer. Prog. Growth Factor Res., /; 84-47, 1484. transformation. 2. Corjay, M. H., Dobrzanski. D. J., Way. J. M.. Viallet. J.. StupirÃ . H.. Worland. P., It is possible that bombesin receptor subtypes might be highly Sausville. E. A., and Battey, J. F. Two distinct bomhcsin receptor subtypes arc expressed in a minority of HBE cells that are sensitive to the [Ca2 *]Â¡- expressed and functional in human lung carcinoma cells. J. Biol. Chem., 266: elevating and growth-enhancing effects of bombesin. Our assays 18771-18779, 1941. 3. Fathi, Z.. Corjay, M. H., Shapira. H.. Wada. E.. Benya, R., Jensen. R., Viallet, J., using mass populations of cells do not allow us to discriminate Sausville. E. A., and Battey, J. F. BRS-3: a novel bombesin receptor subtype selectively expressed in teslis and lung carcinoma cells. J. Biol. C'hem.. 26K: 5474- between this possibility and that of a uniform low expression of 5984, 1993. bombesin receptor subtypes and unresponsiveness to bombesin. How 4. Heikkila. R., Trepel, J. B.. Cullila, F., Neckers, L. M. and Sausville. E. A. Bombcsin- ever, the fact that the BRS-3 subtype is not expressed in HBE cells related peptides induce calcium mohili/alion in a subset of human small cell lung cancer cell lines. J. Biol. Chem.. 262: 16456-16460, 19X7. and that mass cultures of lung cancer cells show higher levels of 5. Trepel, J. B.. Mover. J. D.. Heikkila. R. and Sausville. E. A. 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Our findings are in contrast with previous reports documenting an 8. Willey. J. C.. Lechner, J. F., and Harris, C. C. Bombesin and the C-terminal tetradecapeptide ot gastrin-releasing peptide are growth factors lor normal human enhanced donai growth of some primary HBE cells in response to bronchial epithelial cells. Exp. Cell Res.. 153: 245-24Â«. 14X4. bombesin (8, 9). Siegfried et al. (9) reported recently that bombesin 4. Siegfried. J. M.. Guentert. P. J.. and Gaither. A. L. EÃ®ffeclsof bombcsin and either stimulated (8 of 13) or inhibited (5 of 13) the donai growth of gaslrin-releasing peptide on human bronchial epithelial cells from a series of donors: individual variation and modulation by hombesin analogs. Anat. Ree.. 236: 241-247. primary HBE cells. The lung samples obtained for the primary cul 1993. tures in this report were mostly from donors undergoing pulmonary 10. Tsao, M-S., Zhu, H.. Giaid. A., Viallet. J.. Nakamura. T.. and Park, M. Hepatocyle 1615

growth factor/scatter factor is an autocrine factor for human normal bronchial epi- Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin thelial cells and lung carcinoma cells. Cell Growth & Differ., 4: 571-579, 1993. sensitivity and overall heterogeneity. Cancer Res., 52: 24-31, 1992. 11. Munger. K.. Phelps, W. C., Bubb, V., Howley, P. M., and Schlegel, R. The Â£6and 14. Bunn, P. A. J., Dienhart. D. G., Chan, D., Tagawa, M., and Jewell. P. Effects of Â£7genes of the human papillomavirus type 16 together are necessary and sufficient neuropeptides on human lung and breast cancer cells. J. Nati. Cancer Inst. Monogr.. for transformation of primary human keratinocytes. J. Viral., 63: 4417-4421, 1989. 13: 145-151, 1992. 12. Viallet, J., Liu, C., Emond, J., and Tsao, M-S. Characterization of human bronchial 15. Noguchi, M., Murakami, M., Bennett, W., Lupu, R., Hui, F., Harris, C. C., and epithelial cells immortalized by the Â£6and Â£7genes of human papillomavirus type Gerwin, B. 1. Biological consequences of overexpression of a transfected c-croB-2 16. Exp. Cell Res., in press, 1994. gene in immortalized human bronchial epithelial cells. Cancer Res., 53: 2035-2043, 13. Bunn, P. A. J., Chan, D., Dienhart, D. G., Tolley, R.. Tagawa, M., and Jewett, P. B. 1993.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1994 American Association for Cancer Research. Receptor Subtype Expression and Responsiveness to Bombesin in Cultured Human Bronchial Epithelial Cells

Andrea Frankel, Ming-Sound Tsao and Jean Viallet

Cancer Res 1994;54:1613-1616.

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