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The proliferative response of HT-29 human colon adenocarcinoma cells to bombesin-like peptides
Cancer Letters 172 (2001) 151–157 www.elsevier.com/locate/canlet
The proliferative response of HT-29 human colon adenocarcinoma cells to bombesin-like peptides Giuseppe Cassano a, Nicoletta Resta b, Giuseppe Gasparre a, Claudio Lippe a, Ginevra Guanti b,* b
a Dipartimento di Fisiologia Generale ed Ambientale, Universita` di Bari, Via Amendola 165/A, 70126 Bari, Italy Sez. Genetica medica, Dipartimento di Medicina Interna e Medicina Pubblica, Piazza Giulio Cesare 11, Universita` di Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy
Received 25 April 2001; received in revised form 12 June 2001; accepted 13 June 2001
Abstract Bombesin-like peptides (BLP) and their receptors are widely distributed throughout the intestine and are potential mitogens for gastrointestinal cancers. In this study we characterized the proliferation induced by BLP in the human adenocarcinoma cell line HT-29. The number of HT-29 cells, partially serum deprived (1% fetal bovine serum) for 48 h, was increased after 24 h of stimulation with bombesin, GRP, neuromedin B (NMB) and neuromedin C (NMC) ranging from 0.1 nM up to 1 mM. Reverse transcription polymerase chain reaction studies, revealed the presence of mRNA for NMB and for the GRP preferring receptor (GRP-R). mRNA for GRP, NMB preferring receptor (NMB-R) and bombesin receptor subtype 3 (BRS-3) were not detected. [d-Phe 6]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2), are considered as inhibitors of binding to GRP-R and NMBR, respectively. Surprisingly, A1 and A2 stimulated the proliferation of HT-29 cells. Moreover, in the simultaneous presence of 1 mM A1 and 0.1 mM GRP or 0.1 nM or 0.1 mM bombesin, inhibition of the proliferation was observed. Our data demonstrate that the proliferation induced by BLP in HT-29 cells is due to interaction with the GRP-R. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Colorectal-cancer; Bombesin; Gastrin-releasing-peptide; Neuromedin-B; Neuromedin-C; Reverse-transcription-polymerase-chainreaction
1. Introduction The tetradecapeptide bombesin was originally isolated from the skin of the frog Bombina bombina [1]. Several structurally related peptides were subsequently isolated and classified into three subfamilies (the bombesin, ranatensin and litorin subfamilies)
* Corresponding author. Tel.: 139-080-5478270; fax: 139-080 5478269. E-mail address: [email protected] (G. Guanti).
according to their COOH-terminal tripeptides [2]. At present, three mammalian bombesin-like peptides have been identified: gastrin-releasing peptide (GRP), neuromedin C (NMC, also referred to as GRP-10 because it corresponds to the C-terminal decapeptide of GRP), and neuromedin B (NMB). GRP and NMC, like bombesin, have Leu as the penultimate residue from their C-terminus. NMB, like ranatensin, has Phe instead of Leu as its penultimate residue. No mammalian member of the litorin subfamily has yet been identified. In rodents and humans, three bombesin receptor
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00642-5
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subtypes have been identified: GRP-R or gastrinreleasing peptide preferring receptor [3,4]; NMB-R or neuromedin B preferring receptor [5]; and BRS-3 or bombesin receptor subtype 3, which has a not yet identified high affinity ligand [6,7]. Mammalian bombesin-like peptides induce secretion of hormones, gastric acid and mucous secretion, regulate smooth muscle contraction and modulate the neuronal firing rate [2]. Bombesin-like peptides are also mitogens in cell culture model systems; Rozengurt and Sinnett-Smith [8] demonstrated that bombesin addition to Swiss 3T3 fibroblasts induced DNA synthesis. Although extensive information is available on the role of this family of receptors in various species, relatively few such studies exist for humans. Recent studies show that bombesin-like peptides can function as growth factors in human tumors [2,9]. Interestingly, the GRP-R expressed by the nonmalignant human colon epithelial cell line NCM460 was constitutively active and caused cell proliferation [10]. These results prompted us to investigate the functional state of the receptors of bombesin-like peptides in HT-29 cells and to ascertain whether or not receptor activation leads to proliferation. The HT-29 cell line was originally isolated from human colon adenocarcinoma [11,12] and it is a suitable model for studying the molecular mechanisms of colonic tumorigenesis and the influence of hormonal manipulations on tumor growth. In the first paper dealing with bombesin and HT-29 cells [13] it was reported that ‘growth of HT29 human colon cancer xenografts in nude mice’ was inhibited ‘by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095)’. Interestingly, HT29 cells growing in vitro need to be partially serum deprived (1% fetal bovine serum) to show stimulation of proliferation dependent on bombesin [14]. In this paper we analysed, using a reverse-transcription-polymerase-chain-reaction (RT-PCR) technique, HT-29 cells for the presence of receptors of bombesin-like peptides. Moreover, to get information on the functional state of the receptor, we measured cell proliferation after stimulation with bombesin, GRP, NMC and NMB. Finally, we challenged GRP and bombesin stimulation of proliferation with [DPhe 6]bombesin-(6-13)methyl ester [15] and BIM23127 [16], antagonists of GRP-R and NMB-R, respectively.
2. Materials and methods 2.1. Chemicals Bombesin-like peptides were purchased from Sigma; antagonists were a kind gift from Prof D.H. Coy (Tulane University, New Orleans, LA). These substances were stored as stock solution in the medium used for cell growth, without serum.
2.2. RNA extraction and reverse transcription Total cellular RNA was extracted using the ‘High pure RNA isolation kit’ (Roche). Reverse transcription was performed using 400 U MMLV-RT (Gibco-BRL) in manufacturer’s buffer containing 0.5 mM dNTP, 10 mM dithiothreitol (DDT) and 0.05 unit pdt (12–18) primer (Promega); cDNA integrity was verified by PCR amplification of a fragment of the constitutively expressed b-actin gene (primers and conditions obtained from Stratagene).
2.3. Polymerase chain reaction The primers for GRP-R, GRP ligand, NMB-R, NMB ligand and BRS-3 were as reported by Chave and co-workers [17]. The amplification was performed at 0.25 mM final concentration using 250 mM each of dNTP, 1.0 U Taq Gold (Perkin Elmer) in manufacturer’s buffer containing 2 mM MgCl2 and 2% dimethyl sulfoxide (DMSO) (Sigma) for GRPR; 2.5 mM MgCl2 without DMSO for GRP ligand; 3 mM MgCl2 and 1% DMSO for NMB-R; 1.5 mM MgCl2 and 2% DMSO for NMB ligand and BRS-3. Cycling was carried out [17] on a Hybrid Express thermal cycler with an initial denaturing step of 3 min at 948C followed by 30 cycles of 948C denaturation for 30 s, annealing at 628C (GRP-R); 568C (GRP ligand); 568C (NMB-R); 668C (NMB ligand); 568C (BRS-3) for 30 s and 728C polymerization for 30 s. A final extension step of 728C for 2 min was performed to ensure completion of all initiated polymerization events. The PCR products were characterized on 6% nondenaturing polyacrylamide gel. The bands were visualized by silver staining and photographed.
G. Cassano et al. / Cancer Letters 172 (2001) 151–157
2.4. Cell culture and proliferation assay The human colon adenocarcinoma cell line HT-29 was purchased from the American Type Culture Collection (Rockville, MD). Cell cultures were grown in RPMI 1640 (ICN Flow) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin. Cells were maintained at 378C and under 5% CO2 in humidified air. Subdivision was achieved by exposure to a trypsin/EDTA solution (0.05 %/0.02 %). Cells were seeded into 24-well plates in growth medium containing 10% FBS. After 72 h, the medium was replaced with a fresh medium containing 1% FBS. After a further 48 h, cells were washed with phosphate buffered saline (PBS) and fresh medium, without FBS, and bombesin (or vehicle) was added; after 30 min, 1% (final concentration) FBS was added. After 24 h cells were detached by a trypsin/EDTA solution (0.05/0.02%), resuspended in normal growth medium and counted using a hemocytometer. The data shown are means ^ SEM (whenever it exceeded the size of symbols) of three replicates from three experiments. In Figs. 3 and 4, values have been normalized on the control value (the number of cells in the absence of stimulant and/or antagonist). Data were statistically analyzed with the Student t-test, when indicated.
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been plotted, against time, to make possible the direct visual comparison of different growth rates (slope of the lines interpolating time points). Fig. 1 shows that, as expected, cells proliferated more rapidly in 10% FBS than in 1% FBS; after 72 h in 1% FBS, the cell number was lower than that measured after 48 h and the addition of bombesin restored cell proliferation, although at a lower rate than that observed in 10% FBS. The effect of bombesin on the proliferation of HT29 cells was only observed under the experimental conditions described above and reported in Fig. 1. No effects on the proliferation were obtained when bombesin was added to cells maintained in a medium without FBS or containing 10% FBS, or to cells kept in 1% FBS medium for a period of time of 24 h (and not of 48 h as in Fig. 1). 3.2. RT-PCR studies In order to elucidate the substrate and receptor specificity of the stimulation by bombesin-like peptides of the proliferation in HT-29 cells, we analysed the expression of the mRNA of two bombesin-like peptides (namely GRP and NMB) and their mammalian receptor subtypes (GRP-R, NMB-R and BRS-3). These studies were carried out in HT-29 cells maintained for 48 h in 1% FBS medium, and are reported in Fig. 2. Among mRNA for receptors, only the GRP-R mRNA was detectable. Moreover,
3. Results 3.1. Effect of bombesin on the proliferation of HT-29 cells As shown in Fig. 1, cells were seeded in a growth medium containing 10% FBS; the medium, after 72 h, was replaced by a fresh growth medium containing 1% FBS; after a further 48 h medium was removed and a fresh medium without serum plus 10 nM bombesin was added; after 30 min incubation 1% FBS (final concentration) was added. Cell number is dependent on the time of culture according to the following equation: N ¼ N0 ·elt (where N is the cell number, N0 is the cell number at time zero, t is time and l is a coefficient estimating the growth rate depending on cell type and conditions of culture). In Fig. 1, the logarithm of cell number has
Fig. 1. Stimulation by 10 nM bombesin of the proliferation of HT29 cells. The bars above the graph indicate the concentration of FBS in the medium; interruption of the bar indicates that a medium change was performed. Means ^ SEM were calculated from triplicates from three experiments.
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3.3. Ligand specificity of the GRP-R in HT-29 cells To evaluate the specificity of the GRP-R for ligands in HT-29 cells, we investigated the effect of four bombesin-like peptides (GRP, NMC, NMB and bombesin) between 0.1 nM and 1 mM. This range of concentrations was chosen because Carroll and coworkers [18], in the same cell line, measured a Ki value of 1.9 ^ 0.4 nM (inhibition by bombesin of the 125I-Tyr 4-bombesin binding). The results are reported in Fig. 3. Surprisingly, the proliferation of HT-29 cells was stimulated by all four peptides with a similar pattern of dependence on substrate concentration; the maximal stimulation was reached at 10 nM for all four peptides. We also investigated the potential inhibitory effect of two presumed specific antagonists for the GRP-R and the NMB-R, namely [d-Phe 6]bombesin-(613)methyl ester [15] and BIM-23127 [16], indicated as A1 and A2, respectively. Fig. 4 shows inhibition experiments of the stimulation by 0.1 mM GRP and 0.1 mM bombesin. Both A1 and A2 induced a significant increase in cell number; such an effect was not additive to GRP or bombesin stimulation. Remarkably, the simultaneous presence of 1 mM A1 and 0.1 mM GRP or bombesin lowered the cell number to the level of the control value.
Fig. 2. RT-PCR analysis of the mRNA expression of bombesin-like peptides and their receptors in HT-29 cells. GRP-R, GRP receptor; NMB-R, neuromedin B receptor; BRS-3, bombesin subtype 3 receptor; NMB, neuromedin B. Cells were seeded in a growth medium containing 10% FBS; after 72 h, the medium was replaced with a fresh medium containing 1% FBS and after a further 48 h mRNA was extracted.
NMB mRNA was identified, while GRP mRNA was absent. Therefore, HT-29 cells, under the specific experimental conditions used, did not express mRNA for GRP but expressed mRNA for its specific receptor GRP-R, while the opposite situation was found for NMB (mRNA for the ligand and not for the receptor). These findings indicate that the bombesin effect on proliferation of HT-29 cells is triggered by the exclusive interaction with the GRP-R.
Fig. 3. Stimulation by GRP, neuromedin C, bombesin, and neuromedin B of the proliferation of HT-29 cells. Experimental protocol was identical to that of Fig.1. Means ^ SEM were calculated from normalized triplicates of three experiments, using as 100% the cell number in the wells without stimulants (43556, 46670, 104720, respectively). With the exception of the values measured at 0.1 mM neuromedin C and 1 mM GRP, all data were significantly (P , 0:05) different from controls.
G. Cassano et al. / Cancer Letters 172 (2001) 151–157
The experiments reported in Fig. 4, were then repeated using 0.1 nM GRP and bombesin (not shown). Similar results were obtained with one exception; in the presence of 0.1 nM GRP and 1 mM A1 no inhibition was observed while 0.1 nM bombesin and 1 mM A1 lowered cell number to the level of the control value. All together these results led us to conclude that the two presumed antagonists were able to increase the proliferation of HT-29 cells acting as an agonist.
4. Discussion In this paper we show that HT-29 cells, maintained for 48 h in 1% FBS, proliferate after stimulation with bombesin; our findings confirm the results obtained by Casanueva and co-workers [14]. Under similar experimental conditions, GRP, NMC, and NMB too induced the proliferation of HT-29 cells. With RT-PCR we demonstrated that HT-29 cells, maintained in 1% FBS, only express GRP-R and NMB, among receptors and bombesin-like peptides, respectively. Carroll and co-workers [18] found both GRP-R and GRP ligand mRNAs in HT-29 cells, as in several other human colon cancer cell lines; we suggest that such a discrepancy is due to the fact that the two observations were made using cells kept under different growing conditions (1% FBS in our report and 10% in the other case). The expression
Fig. 4. Effect of [d-Phe 6]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2) on the proliferation of HT-29 cells induced by GRP and bombesin. *Significantly (P , 0:05) different from control (100%). Means ^ SEM were calculated from normalized triplicates of six (open bars) or three (dashed bars) experiments, using as 100% the cell number in the wells without stimulants (34483, 91556, 69692 and 30010, 40089, 58889 for GRP and bombesin experiments, respectively).
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of NMB mRNA rises the question whether NMB may act in an autocrine loop to promote proliferation. This is not the case of HT-29 cells, because in the absence of FBS, we did not measure increase of cell proliferation. [d-Phe 6]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2) are commonly considered as antagonists of GRP-R and NMB-R respectively; surprisingly, in HT-29 cells these molecules stimulated proliferation, in the absence of other peptides. Nevertheless, in three cases (1 mM A1 1 either 0.1 mM GRP or 0.1 nM bombesin or 0.1 mM bombesin), inhibition of stimulation was observed; this effect could be possibly explained as due to the receptor desensitization and the fact that it was only observed when A1 was used, confirms that GRP-R is functional in HT-29 cells. In our experiments we obtained two unexpected results: the proliferation of HT-29 cells was stimulated by all four peptides with a similar pattern of dependence on substrate concentration and two presumed antagonists promoted proliferation. Taken together, this experimental evidence could suggest that the GRP-R, of the HT-29 cells used in our study, is mutated. On the other hand, Carroll and co-workers [18] have demonstrated that the mutations, present in the GRP-R of HT-29 cells, are silent. In our opinion, our data could be caused by one or more post-translational modifications, that are altered in HT-29 cells, and that occur in the other cell types used to investigate the role played by GRP-R. The importance of post-translational processes has already been demonstrated by a study about the ‘glycosylation of the gastrin-releasing peptide receptor and its effect on expression, G protein coupling, and receptor modulatory processes’, in transfected CHOP cells [19]. The expression of the GRP and GRP-R genes in colorectal cancer cells is extremely variable. Saurin and co-workers [20] analysed 29 surgical tumor specimens with RT-PCR and found that 27 samples (93%) express GRP-R mRNA. In another study [17], samples from colorectal cancer tissue and matched normal mucosa from 23 patients were analysed. In this latter case the GRP receptor and ligand expression was found in all samples with a greater expression in the tumor samples. On the other hand, tumors express GRP-R mRNA more frequently than functional protein. In fact, Radu-
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lovic and co-workers [21] and Preston and co-workers [9], using 125I-Tyr 4-bombesin, observed the presence of functional receptors in 6 out of 15 and in 5 out of 21 tumors, respectively; conversely, Carroll and coworkers [22] demonstrated the presence of GRP-R protein in 76% of tumors by immunohistochemistry. A recent paper has proposed that mutations of genes coding for GRP-R receptors account for the failure of functional protein to be generated [18]. HT-29 is one of the cell lines most frequently used in studies investigating the mechanisms of activation of the intracellular processes inducing proliferation. This work indicates that HT-29 cells have a functional GRP-R the activation of which induces cell proliferation. For this reason HT-29 cells represent a useful experimental tool to evaluate not just the expression of the mRNA, but also the existence of functional receptors with their connection to the intracellular mechanisms of signal transduction. Acknowledgements This work was supported by grants from the University of Bari, from ‘Ministero della Ricerca Scientifica e Tecnologica’ Cofin ‘98, and from AIRC ‘Progetto tumori ereditari del colon’. References [1] A. Anastasi, V. Erspamer, M. Bucci, Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphybians bombina and alytes, Experientia 27 (1971) 166–167. [2] G.S. Kroog, R. Jensen, J.F. Battey, Mammalian bombesin receptors, Med. Res. Rev. 15 (1995) 389–417. [3] E.R. Spindel, E. Giladi, P. Brehm, R.H. Goodman, T.P. Segerson, Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrinreleasing peptide receptor, Mol. Endocrinol. 4 (1990) 1956– 1963. [4] J.F. Battey, J.M. Way, M.H. Corjay, H. Shapira, K. Kusano, R. Harkins, J.M. Wu, T. Slattery, E. Mann, R.I. Feldman, Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 395–399. [5] E. Wada, J. Way, H. Shapira, K. Kusano, A.M. LebacqVerheyden, D. Coy, R. Jensen, J. Battey, cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor, Neuron 6 (1991) 421– 430.
[6] V. Gorbulev, A. Akhundova, H. Buchner, F. Fahrenholz, Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy, Eur. J. Biochem. 208 (1992) 405–410. [7] Z. Fathi, M.H. Corjay, H. Shapira, E. Wada, R. Benya, R. Jensen, J. Viallet, E.A. Suasville, J.F. Battey, BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells, J. Biol. Chem. 268 (1993) 5979–5984. [8] E. Rozengurt, J. Sinnett-Smith, Bombesin stimulation of DNA synthesis and cell division in cultures of Swiss 3T3 cells, Proc. Natl. Acad. Sci. 80 (1983) 2936–2940. [9] S.R. Preston, G.V. Miller, J.N. Primrose, Bombesin-like peptides and cancer, Crit. Rev. Oncol. Hematol. 23 (1996) 225–238. [10] H.A. Ferris, R.E. Carroll, M.M. Rasenick, R.V. Benya, Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460, J. Clin. Invest. 100 (1997) 2530–2537. [11] M. Rousset, The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation, Biochimie 68 (1986) 1035–1040. [12] D.L. Trainer, T. Kline, F. McCabe, L.F. Faucette, J. Feild, M. Chaikin, M. Anzano, D. Rieman, S. Hoffstein, D.J. Li, D. Gennaro, C. Buscarino, M. Lynch, G. Poste, R. Greig, Biological characterization and oncogene expression in human colorectal carcinoma cell lines, Int. J. Cancer 41 (1988) 287–296. [13] S. Radulovic, G. Miller, A.V. Schally, Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC3095), Cancer Res. 51 (1991) 6006–6009. [14] F.F. Casanueva, F.R. Perez, X. Casabiell, J. Camina, R.Z. Cai, A.V. Schally, Correlation between the effects of bombesin antagonists on cell proliferation and intracellular calcium concentration in Swiss 3T3 and HT-29 cell lines, Proc. Natl. Acad. Sci U.S.A. 93 (1996) 1406–1411. [15] R.V. Benya, T. Kusui, T.K. Pradhan, J.F. Battey, R.T. Jensen, Expression and characterization of cloned human bombesin receptors, Mol. Pharmacol. 47 (1995) 10–20. [16] E.E. Ladenheim, J.E. Taylor, D.H. Coy, T.H. Moran, Blockade of feeding inhibition by neuromedin B using a selective receptor antagonist, Eur. J. Pharmacol. 271 (1994) R7–R9. [17] H.S. Chave, A.C. Gough, K. Palmer, S.R. Preston, J.N. Primrose, Bombesin family receptor and ligand gene expression in human colorectal cancer and normal mucosa, Br. J. Cancer 82 (2000) 124–130. [18] R.E. Carroll, D. Ostrovskiy, S. Lee, A. Danilkovich, R.V. Benya, Characterization of gastrin-releasing peptide and its receptor aberrantly expressed by human colon cancer cell lines, Mol. Pharmacol. 58 (2000) 601–607. [19] R.V. Benya, T. Kusui, T. Katsuno, T. Tsuda, S.A. Mantey, J.F. Battey, R.T. Jensen, Glycosylation of the gastrin-releasing peptide receptor and its effect on expression, G protein coupling, and receptor modulatory processes, Mol. Pharmacol. 58 (2000) 1490–1501. [20] J.C. Saurin, J.P. Rouault, J. Abello, F. Berger, L. Remy, J.A.
G. Cassano et al. / Cancer Letters 172 (2001) 151–157 Chayvialle, High gastrin releasing peptide receptor mRNA level is related to tumour dedifferentiation and lymphatic vessel invasion in human colon cancer, Eur. J. Cancer 35 (1999) 125–132. [21] S.S. Radulovic, S.R. Milovanovic, R.Z. Cai, A.V. Schally, The binding of bombesin and somatostatin and their analogs
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to human colon cancers, Proc. Soc. Exp. Biol. Med. 200 (1992) 394–401. [22] R.E. Carroll, K.A. Matkowskyj, S. Chakrabarti, T.J. McDonald, R.V. Benya, Aberrant expression of gastrin-releasing peptide by well differentiated colon cancers in humans, Am. J. Physiol. 276 (1999) G655–G665.
The proliferative response of HT-29 human colon adenocarcinoma cells to bombesin-like peptides Giuseppe Cassano a, Nicoletta Resta b, Giuseppe Gasparre a, Claudio Lippe a, Ginevra Guanti b,* b
a Dipartimento di Fisiologia Generale ed Ambientale, Universita` di Bari, Via Amendola 165/A, 70126 Bari, Italy Sez. Genetica medica, Dipartimento di Medicina Interna e Medicina Pubblica, Piazza Giulio Cesare 11, Universita` di Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy
Received 25 April 2001; received in revised form 12 June 2001; accepted 13 June 2001
Abstract Bombesin-like peptides (BLP) and their receptors are widely distributed throughout the intestine and are potential mitogens for gastrointestinal cancers. In this study we characterized the proliferation induced by BLP in the human adenocarcinoma cell line HT-29. The number of HT-29 cells, partially serum deprived (1% fetal bovine serum) for 48 h, was increased after 24 h of stimulation with bombesin, GRP, neuromedin B (NMB) and neuromedin C (NMC) ranging from 0.1 nM up to 1 mM. Reverse transcription polymerase chain reaction studies, revealed the presence of mRNA for NMB and for the GRP preferring receptor (GRP-R). mRNA for GRP, NMB preferring receptor (NMB-R) and bombesin receptor subtype 3 (BRS-3) were not detected. [d-Phe 6]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2), are considered as inhibitors of binding to GRP-R and NMBR, respectively. Surprisingly, A1 and A2 stimulated the proliferation of HT-29 cells. Moreover, in the simultaneous presence of 1 mM A1 and 0.1 mM GRP or 0.1 nM or 0.1 mM bombesin, inhibition of the proliferation was observed. Our data demonstrate that the proliferation induced by BLP in HT-29 cells is due to interaction with the GRP-R. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Colorectal-cancer; Bombesin; Gastrin-releasing-peptide; Neuromedin-B; Neuromedin-C; Reverse-transcription-polymerase-chainreaction
1. Introduction The tetradecapeptide bombesin was originally isolated from the skin of the frog Bombina bombina [1]. Several structurally related peptides were subsequently isolated and classified into three subfamilies (the bombesin, ranatensin and litorin subfamilies)
* Corresponding author. Tel.: 139-080-5478270; fax: 139-080 5478269. E-mail address: [email protected] (G. Guanti).
according to their COOH-terminal tripeptides [2]. At present, three mammalian bombesin-like peptides have been identified: gastrin-releasing peptide (GRP), neuromedin C (NMC, also referred to as GRP-10 because it corresponds to the C-terminal decapeptide of GRP), and neuromedin B (NMB). GRP and NMC, like bombesin, have Leu as the penultimate residue from their C-terminus. NMB, like ranatensin, has Phe instead of Leu as its penultimate residue. No mammalian member of the litorin subfamily has yet been identified. In rodents and humans, three bombesin receptor
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00642-5
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subtypes have been identified: GRP-R or gastrinreleasing peptide preferring receptor [3,4]; NMB-R or neuromedin B preferring receptor [5]; and BRS-3 or bombesin receptor subtype 3, which has a not yet identified high affinity ligand [6,7]. Mammalian bombesin-like peptides induce secretion of hormones, gastric acid and mucous secretion, regulate smooth muscle contraction and modulate the neuronal firing rate [2]. Bombesin-like peptides are also mitogens in cell culture model systems; Rozengurt and Sinnett-Smith [8] demonstrated that bombesin addition to Swiss 3T3 fibroblasts induced DNA synthesis. Although extensive information is available on the role of this family of receptors in various species, relatively few such studies exist for humans. Recent studies show that bombesin-like peptides can function as growth factors in human tumors [2,9]. Interestingly, the GRP-R expressed by the nonmalignant human colon epithelial cell line NCM460 was constitutively active and caused cell proliferation [10]. These results prompted us to investigate the functional state of the receptors of bombesin-like peptides in HT-29 cells and to ascertain whether or not receptor activation leads to proliferation. The HT-29 cell line was originally isolated from human colon adenocarcinoma [11,12] and it is a suitable model for studying the molecular mechanisms of colonic tumorigenesis and the influence of hormonal manipulations on tumor growth. In the first paper dealing with bombesin and HT-29 cells [13] it was reported that ‘growth of HT29 human colon cancer xenografts in nude mice’ was inhibited ‘by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095)’. Interestingly, HT29 cells growing in vitro need to be partially serum deprived (1% fetal bovine serum) to show stimulation of proliferation dependent on bombesin [14]. In this paper we analysed, using a reverse-transcription-polymerase-chain-reaction (RT-PCR) technique, HT-29 cells for the presence of receptors of bombesin-like peptides. Moreover, to get information on the functional state of the receptor, we measured cell proliferation after stimulation with bombesin, GRP, NMC and NMB. Finally, we challenged GRP and bombesin stimulation of proliferation with [DPhe 6]bombesin-(6-13)methyl ester [15] and BIM23127 [16], antagonists of GRP-R and NMB-R, respectively.
2. Materials and methods 2.1. Chemicals Bombesin-like peptides were purchased from Sigma; antagonists were a kind gift from Prof D.H. Coy (Tulane University, New Orleans, LA). These substances were stored as stock solution in the medium used for cell growth, without serum.
2.2. RNA extraction and reverse transcription Total cellular RNA was extracted using the ‘High pure RNA isolation kit’ (Roche). Reverse transcription was performed using 400 U MMLV-RT (Gibco-BRL) in manufacturer’s buffer containing 0.5 mM dNTP, 10 mM dithiothreitol (DDT) and 0.05 unit pdt (12–18) primer (Promega); cDNA integrity was verified by PCR amplification of a fragment of the constitutively expressed b-actin gene (primers and conditions obtained from Stratagene).
2.3. Polymerase chain reaction The primers for GRP-R, GRP ligand, NMB-R, NMB ligand and BRS-3 were as reported by Chave and co-workers [17]. The amplification was performed at 0.25 mM final concentration using 250 mM each of dNTP, 1.0 U Taq Gold (Perkin Elmer) in manufacturer’s buffer containing 2 mM MgCl2 and 2% dimethyl sulfoxide (DMSO) (Sigma) for GRPR; 2.5 mM MgCl2 without DMSO for GRP ligand; 3 mM MgCl2 and 1% DMSO for NMB-R; 1.5 mM MgCl2 and 2% DMSO for NMB ligand and BRS-3. Cycling was carried out [17] on a Hybrid Express thermal cycler with an initial denaturing step of 3 min at 948C followed by 30 cycles of 948C denaturation for 30 s, annealing at 628C (GRP-R); 568C (GRP ligand); 568C (NMB-R); 668C (NMB ligand); 568C (BRS-3) for 30 s and 728C polymerization for 30 s. A final extension step of 728C for 2 min was performed to ensure completion of all initiated polymerization events. The PCR products were characterized on 6% nondenaturing polyacrylamide gel. The bands were visualized by silver staining and photographed.
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2.4. Cell culture and proliferation assay The human colon adenocarcinoma cell line HT-29 was purchased from the American Type Culture Collection (Rockville, MD). Cell cultures were grown in RPMI 1640 (ICN Flow) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin. Cells were maintained at 378C and under 5% CO2 in humidified air. Subdivision was achieved by exposure to a trypsin/EDTA solution (0.05 %/0.02 %). Cells were seeded into 24-well plates in growth medium containing 10% FBS. After 72 h, the medium was replaced with a fresh medium containing 1% FBS. After a further 48 h, cells were washed with phosphate buffered saline (PBS) and fresh medium, without FBS, and bombesin (or vehicle) was added; after 30 min, 1% (final concentration) FBS was added. After 24 h cells were detached by a trypsin/EDTA solution (0.05/0.02%), resuspended in normal growth medium and counted using a hemocytometer. The data shown are means ^ SEM (whenever it exceeded the size of symbols) of three replicates from three experiments. In Figs. 3 and 4, values have been normalized on the control value (the number of cells in the absence of stimulant and/or antagonist). Data were statistically analyzed with the Student t-test, when indicated.
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been plotted, against time, to make possible the direct visual comparison of different growth rates (slope of the lines interpolating time points). Fig. 1 shows that, as expected, cells proliferated more rapidly in 10% FBS than in 1% FBS; after 72 h in 1% FBS, the cell number was lower than that measured after 48 h and the addition of bombesin restored cell proliferation, although at a lower rate than that observed in 10% FBS. The effect of bombesin on the proliferation of HT29 cells was only observed under the experimental conditions described above and reported in Fig. 1. No effects on the proliferation were obtained when bombesin was added to cells maintained in a medium without FBS or containing 10% FBS, or to cells kept in 1% FBS medium for a period of time of 24 h (and not of 48 h as in Fig. 1). 3.2. RT-PCR studies In order to elucidate the substrate and receptor specificity of the stimulation by bombesin-like peptides of the proliferation in HT-29 cells, we analysed the expression of the mRNA of two bombesin-like peptides (namely GRP and NMB) and their mammalian receptor subtypes (GRP-R, NMB-R and BRS-3). These studies were carried out in HT-29 cells maintained for 48 h in 1% FBS medium, and are reported in Fig. 2. Among mRNA for receptors, only the GRP-R mRNA was detectable. Moreover,
3. Results 3.1. Effect of bombesin on the proliferation of HT-29 cells As shown in Fig. 1, cells were seeded in a growth medium containing 10% FBS; the medium, after 72 h, was replaced by a fresh growth medium containing 1% FBS; after a further 48 h medium was removed and a fresh medium without serum plus 10 nM bombesin was added; after 30 min incubation 1% FBS (final concentration) was added. Cell number is dependent on the time of culture according to the following equation: N ¼ N0 ·elt (where N is the cell number, N0 is the cell number at time zero, t is time and l is a coefficient estimating the growth rate depending on cell type and conditions of culture). In Fig. 1, the logarithm of cell number has
Fig. 1. Stimulation by 10 nM bombesin of the proliferation of HT29 cells. The bars above the graph indicate the concentration of FBS in the medium; interruption of the bar indicates that a medium change was performed. Means ^ SEM were calculated from triplicates from three experiments.
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3.3. Ligand specificity of the GRP-R in HT-29 cells To evaluate the specificity of the GRP-R for ligands in HT-29 cells, we investigated the effect of four bombesin-like peptides (GRP, NMC, NMB and bombesin) between 0.1 nM and 1 mM. This range of concentrations was chosen because Carroll and coworkers [18], in the same cell line, measured a Ki value of 1.9 ^ 0.4 nM (inhibition by bombesin of the 125I-Tyr 4-bombesin binding). The results are reported in Fig. 3. Surprisingly, the proliferation of HT-29 cells was stimulated by all four peptides with a similar pattern of dependence on substrate concentration; the maximal stimulation was reached at 10 nM for all four peptides. We also investigated the potential inhibitory effect of two presumed specific antagonists for the GRP-R and the NMB-R, namely [d-Phe 6]bombesin-(613)methyl ester [15] and BIM-23127 [16], indicated as A1 and A2, respectively. Fig. 4 shows inhibition experiments of the stimulation by 0.1 mM GRP and 0.1 mM bombesin. Both A1 and A2 induced a significant increase in cell number; such an effect was not additive to GRP or bombesin stimulation. Remarkably, the simultaneous presence of 1 mM A1 and 0.1 mM GRP or bombesin lowered the cell number to the level of the control value.
Fig. 2. RT-PCR analysis of the mRNA expression of bombesin-like peptides and their receptors in HT-29 cells. GRP-R, GRP receptor; NMB-R, neuromedin B receptor; BRS-3, bombesin subtype 3 receptor; NMB, neuromedin B. Cells were seeded in a growth medium containing 10% FBS; after 72 h, the medium was replaced with a fresh medium containing 1% FBS and after a further 48 h mRNA was extracted.
NMB mRNA was identified, while GRP mRNA was absent. Therefore, HT-29 cells, under the specific experimental conditions used, did not express mRNA for GRP but expressed mRNA for its specific receptor GRP-R, while the opposite situation was found for NMB (mRNA for the ligand and not for the receptor). These findings indicate that the bombesin effect on proliferation of HT-29 cells is triggered by the exclusive interaction with the GRP-R.
Fig. 3. Stimulation by GRP, neuromedin C, bombesin, and neuromedin B of the proliferation of HT-29 cells. Experimental protocol was identical to that of Fig.1. Means ^ SEM were calculated from normalized triplicates of three experiments, using as 100% the cell number in the wells without stimulants (43556, 46670, 104720, respectively). With the exception of the values measured at 0.1 mM neuromedin C and 1 mM GRP, all data were significantly (P , 0:05) different from controls.
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The experiments reported in Fig. 4, were then repeated using 0.1 nM GRP and bombesin (not shown). Similar results were obtained with one exception; in the presence of 0.1 nM GRP and 1 mM A1 no inhibition was observed while 0.1 nM bombesin and 1 mM A1 lowered cell number to the level of the control value. All together these results led us to conclude that the two presumed antagonists were able to increase the proliferation of HT-29 cells acting as an agonist.
4. Discussion In this paper we show that HT-29 cells, maintained for 48 h in 1% FBS, proliferate after stimulation with bombesin; our findings confirm the results obtained by Casanueva and co-workers [14]. Under similar experimental conditions, GRP, NMC, and NMB too induced the proliferation of HT-29 cells. With RT-PCR we demonstrated that HT-29 cells, maintained in 1% FBS, only express GRP-R and NMB, among receptors and bombesin-like peptides, respectively. Carroll and co-workers [18] found both GRP-R and GRP ligand mRNAs in HT-29 cells, as in several other human colon cancer cell lines; we suggest that such a discrepancy is due to the fact that the two observations were made using cells kept under different growing conditions (1% FBS in our report and 10% in the other case). The expression
Fig. 4. Effect of [d-Phe 6]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2) on the proliferation of HT-29 cells induced by GRP and bombesin. *Significantly (P , 0:05) different from control (100%). Means ^ SEM were calculated from normalized triplicates of six (open bars) or three (dashed bars) experiments, using as 100% the cell number in the wells without stimulants (34483, 91556, 69692 and 30010, 40089, 58889 for GRP and bombesin experiments, respectively).
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of NMB mRNA rises the question whether NMB may act in an autocrine loop to promote proliferation. This is not the case of HT-29 cells, because in the absence of FBS, we did not measure increase of cell proliferation. [d-Phe 6]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2) are commonly considered as antagonists of GRP-R and NMB-R respectively; surprisingly, in HT-29 cells these molecules stimulated proliferation, in the absence of other peptides. Nevertheless, in three cases (1 mM A1 1 either 0.1 mM GRP or 0.1 nM bombesin or 0.1 mM bombesin), inhibition of stimulation was observed; this effect could be possibly explained as due to the receptor desensitization and the fact that it was only observed when A1 was used, confirms that GRP-R is functional in HT-29 cells. In our experiments we obtained two unexpected results: the proliferation of HT-29 cells was stimulated by all four peptides with a similar pattern of dependence on substrate concentration and two presumed antagonists promoted proliferation. Taken together, this experimental evidence could suggest that the GRP-R, of the HT-29 cells used in our study, is mutated. On the other hand, Carroll and co-workers [18] have demonstrated that the mutations, present in the GRP-R of HT-29 cells, are silent. In our opinion, our data could be caused by one or more post-translational modifications, that are altered in HT-29 cells, and that occur in the other cell types used to investigate the role played by GRP-R. The importance of post-translational processes has already been demonstrated by a study about the ‘glycosylation of the gastrin-releasing peptide receptor and its effect on expression, G protein coupling, and receptor modulatory processes’, in transfected CHOP cells [19]. The expression of the GRP and GRP-R genes in colorectal cancer cells is extremely variable. Saurin and co-workers [20] analysed 29 surgical tumor specimens with RT-PCR and found that 27 samples (93%) express GRP-R mRNA. In another study [17], samples from colorectal cancer tissue and matched normal mucosa from 23 patients were analysed. In this latter case the GRP receptor and ligand expression was found in all samples with a greater expression in the tumor samples. On the other hand, tumors express GRP-R mRNA more frequently than functional protein. In fact, Radu-
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lovic and co-workers [21] and Preston and co-workers [9], using 125I-Tyr 4-bombesin, observed the presence of functional receptors in 6 out of 15 and in 5 out of 21 tumors, respectively; conversely, Carroll and coworkers [22] demonstrated the presence of GRP-R protein in 76% of tumors by immunohistochemistry. A recent paper has proposed that mutations of genes coding for GRP-R receptors account for the failure of functional protein to be generated [18]. HT-29 is one of the cell lines most frequently used in studies investigating the mechanisms of activation of the intracellular processes inducing proliferation. This work indicates that HT-29 cells have a functional GRP-R the activation of which induces cell proliferation. For this reason HT-29 cells represent a useful experimental tool to evaluate not just the expression of the mRNA, but also the existence of functional receptors with their connection to the intracellular mechanisms of signal transduction. Acknowledgements This work was supported by grants from the University of Bari, from ‘Ministero della Ricerca Scientifica e Tecnologica’ Cofin ‘98, and from AIRC ‘Progetto tumori ereditari del colon’. References [1] A. Anastasi, V. Erspamer, M. Bucci, Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphybians bombina and alytes, Experientia 27 (1971) 166–167. [2] G.S. Kroog, R. Jensen, J.F. Battey, Mammalian bombesin receptors, Med. Res. Rev. 15 (1995) 389–417. [3] E.R. Spindel, E. Giladi, P. Brehm, R.H. Goodman, T.P. Segerson, Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrinreleasing peptide receptor, Mol. Endocrinol. 4 (1990) 1956– 1963. [4] J.F. Battey, J.M. Way, M.H. Corjay, H. Shapira, K. Kusano, R. Harkins, J.M. Wu, T. Slattery, E. Mann, R.I. Feldman, Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 395–399. [5] E. Wada, J. Way, H. Shapira, K. Kusano, A.M. LebacqVerheyden, D. Coy, R. Jensen, J. Battey, cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor, Neuron 6 (1991) 421– 430.
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