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Originally published In Press as doi:10.1074/jbc.M005754200 on July 25, 2000

J. Biol. Chem., Vol. 275, Issue 41, 32122-32128, October 13, 2000
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Human Colorectal Cancers Express a Constitutively Active Cholecystokinin-B/Gastrin Receptor That Stimulates Cell Growth*

Mark R. HellmichDagger §, Xian-Liang RuiDagger , Helen L. Hellmich||, R. Y. Declan FlemingDagger , B. Mark EversDagger , and Courtney M. Townsend Jr.Dagger

From the Departments of Dagger  Surgery, § Physiology and Biophysics, and || Internal Medicine, the University of Texas Medical Branch, Galveston, Texas 77555

Received for publication, June 30, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although ectopic expression of the cholecystokinin B/gastrin receptor (CCK-BR) is widely reported in human colorectal cancers, its role in mediating the proliferative effects of gastrin1-17 (G-17) on these cancers is unknown. Here we report the isolation of a novel splice variant of CCK-BR that exhibits constitutive (ligand-independent) activation of pathways regulating intracellular free Ca2+ ([Ca2+]i) and cell growth. The splice variant (designated CCK-BRi4sv for intron 4-containing splice variant) is expressed in colorectal cancers but not in normal colonic mucosa adjacent to the cancer. Balb3T3 cells expressing CCK-BRi4sv exhibited spontaneous, ligand-independent, oscillatory increases in [Ca2+]i, whereas cells expressing wild-type CCK-BR did not. Primary cultures of cells isolated from resected colorectal cancers also exhibited a similar pattern of spontaneous [Ca2+]i oscillations. For both Balb3T3 and primary tumor cells, application of G-17 (10 and 200 nM, respectively) caused an increase in [Ca2+]i. Selective CCK-BR antagonists blocked the G-17-stimulated Ca2+ responses but not the spontaneous [Ca2+]i oscillations. Cells expressing CCK-BRi4sv exhibited an increased growth rate (~2.5-fold), in the absence of G-17, compared with cells expressing wild-type CCK-BR. The selective pattern of expression, constitutive activity, and trophic action associated with CCK-BRi4sv suggest that this variant may regulate colorectal cancer cell proliferation though a gastrin-independent mechanism.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Colorectal cancers are the third leading cause of cancer deaths in the United States (1). It is estimated that approximately 130,000 new cases of colorectal cancer will be diagnosed in the United States this year. Despite major advances in uncovering the basic biochemical and genetic alterations involved in the development and progression of colorectal cancers (2), currently, treatment of this disease still relies predominantly upon surgical resection. Prognosis for survival is determined primarily by stage of disease at the time of diagnosis. Since the majority of patients with colorectal cancers have serosal penetration and nodal involvement at the time of operation, more effective adjuvant therapies are required. A better understanding of the molecular mechanisms regulating colorectal cancer cell proliferation would greatly facilitate the development of novel therapeutic agents.

In addition to regulating gastric acid secretion, the peptide hormone, gastrin (G-17), and its non-amidated precursor, glycine-extended gastrin (G-Gly), stimulate the growth of some colorectal cancers (3-6). The growth-promoting effects of these peptides have been reported in vivo using human colon cancer xenografts (4, 7) and in vitro in various cell lines derived from human colorectal cancers (8). The cholecystokinin B/gastrin receptor (CCK-BR),1 a member of the G protein-coupled receptor superfamily (9, 10), mediates many of the biological actions of G-17, including stimulation of gastric acid secretion by parietal cells in the oxyntic mucosa (11) and enterochromaffin-like cell proliferation (12). Although ectopic expression of CCK-BR has been widely reported in colorectal cancers (13, 14), a role for CCK-BR in mediating the trophic effects of G-17 and related peptides on colorectal cancer cell growth remains controversial. The controversy is due, in part, to variability in the reported prevalence of CCK-BR expression in colorectal cancers (12, 15, 16) and to the observations that the receptor(s) mediating the mitogenic effects of G-17 and G-Gly does not always satisfy the biochemical and pharmacokinetic criteria of CCK-BR (17, 18).

The third cytoplasmic loop domain of G protein-coupled receptors (GPCRs) interacts with heterotrimeric G proteins and for many receptors, including CCK-BR, plays a critical role in the activation of intracellular signal transduction cascades (19), the regulation of ligand binding affinity, and agonist-induced receptor desensitization (20, 21). Because of the importance of the third cytoplasmic loop in regulating the biochemical and pharmacokinetic properties of GPCRs, we examined this region of CCK-BR, expressed in human colorectal cancers, to determine whether mutations existed that could account for some of the reported inconsistencies in the G-17-induced responses. Here we report the identification and isolation of a novel splice variant of the human CCK-BR that is generated by intron 4 retention during RNA processing. The resulting receptor protein contains 69 additional amino acid residues in its third intracellular loop domain. Expression of the novel splice variant (designated CCK-BRi4sv for intron 4-containing splice variant) was detected in human colorectal cancers and adenomatous polyps but not in the normal colonic mucosa adjacent to the cancer. In addition to having ligand binding properties distinct from the previously characterized wild-type CCK-BR (CCK-BRwt), CCK-BRi4sv exhibits constitutive (agonist-independent) activation of pathways regulating the levels of intracellular free Ca2+ ([Ca2+]i) and cell proliferation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Library Construction and Screening-- CCK-BRi4sv was isolated from a cDNA library constructed with double-selected poly(A)+ mRNA from a freshly resected human colon cancer. The cDNA library was constructed using the ZAP Express XL Library Construction Kit (Stratagene, La Jolla, CA). Double-stranded cDNA, ranging in size from 2 to 4 kb, was size-selected on a low melting point agarose gel and ligated into the ZAP Express XL Vector. One million plaques were screened by plaque hybridization using a random-primed [32P]dATP-labeled CCK-BR cDNA probe as described previously (22). After hybridization overnight at 37 °C, the nitrocellulose filter was washed three times (20 min each) in 0.1× SSC and 0.1% SDS at 65 °C and exposed to x-ray film. Plasmid DNA was recovered from the positive phagemids by an in vivo excision reaction. Two cDNA clones of approximately 2.2 kb in length were identified. DNA sequence was determined using an automated DNA sequencer (Applied Biosystems Inc., PRISM). Both clones contained the full-length CCK-BR in which intron 4 was retained. The total nucleotide sequence (2154 bp) of CCK-BRi4sv was submitted to the GenBankTM data base (accession number AF239668).

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)-- Total RNA was isolated using Ultraspec RNA Isolation System (Biotec Laboratories, Inc., Houston, TX) and treated with 1 unit of RNase-free DNase I at 37 °C for 30 min (Promega, Madison, WI). Poly(A)+ mRNA was isolated using Poly "A" Quick mRNA Isolation Kit (Stratagene, La Jolla, CA). 100 ng of mRNA was converted to cDNA using SuperScript II reverse transcriptase (Life Technologies, Inc.) according to previously described methods (23). PCR was performed using 2.5 µl of the RT reaction and the following primers: sense primer, 5'-GCTTTCGCTCTTGTTCTTCATC-3', and antisense primer, 5'-AACGATCACCAGCAACATTCGC-3', in a total volume of 100 µl. The PCR conditions were 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 1 min. A 1-kb DNA ladder (10 µl) (Life Technologies, Inc.) was used to determine the relative size of the PCR products.

RNase Protection Assay-- Plasmid DNA, containing a 185-bp fragment of intron 4, was linearized with HindIII and used as template for labeling of an antisense RNA probe with MAXIscript in vitro transcription kit (Ambion, Austin, TX). Total RNA (50 µg) isolated from colorectal tumors and, patient-match, normal mucosa adjacent to the tumor were hybridized with a [32P]UTP-labeled antisense riboprobe (~4 × 105 cpm). Following digestion with RNases, the protected RNA fragments were separated by electrophoresis (6% polyacrylamide gel) and visualized by autoradiography.

Transfection Technique-- Balb3T3 cells were plated at a density of 8 × 105 cells/100-mm dish in DMEM supplemented with 5% FBS. After 24 h at 37 °C, the cells were transfected with 1-6 µg of either CCK-BRwt, CCK-BRi4sv, or CCK-BRi4sv* plasmid DNA using a 1:2.3 ratio of DNA to FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals). Total RNA was extracted 24 h after transfection using the Ultraspec RNA Isolation System. To eliminate chromosomal DNA contamination, total RNA was treated with RNase-free DNase I, at 37 °C for 30 min.

Competition Binding Studies-- Balb3T3 cells were plated in 12-well plates, initial density of 3 × 104 cells per well, in 2 ml of DMEM supplemented with 5% FBS. After 24 h, the cells were transfected with plasmid DNA containing either CCK-BRi4sv* or CCK-BRwt cDNA as described above. Binding experiments were performed 24 and 48 h after transfection. For competition analyses, cells were washed twice with 1 ml of binding buffer (1× Hank's balanced salt solution, 10 mM HEPES, pH 7.4, 0.1% BSA) and then incubated (1 h at 30 °C) in binding buffer containing 0.05 nM 125I-labeled G-17 (specific activity = 2200 Ci/mmol, Amersham Pharmacia Biotech) and various concentrations of unlabeled competitors (1 pM to 10 µM). The binding reaction was terminated by the addition of 1 ml of ice-cold binding buffer. The cells were scraped from the wells, transferred to an ice-cold glass tube, and spun at 230 × g for 4 min (4 °C). The resulting cell pellets were counted in a Cobra II gamma counter (Parkard Instrument Co.). Total binding averaged approximately 6% of the total counts added to the assays for each receptor. Nonspecific binding was defined as the amount of radioactivity detected in the presence of 10 µM unlabeled G-17. Each data point was determined in triplicate, and the graphs represent the mean ± S.E. of four independent experiments.

Preparation of Primary Tumor Cells and Calcium Imaging-- Tumor tissue was collected immediately following resection and placed into a sterile tube contain DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Tissue was minced in 1 ml of DMEM plus FBS and antibiotic. After mincing, the tissue was placed into a 15-ml falcon tube containing 2 ml of media supplemented with 1000 units/ml collagenase II (Sigma), incubated for 10 min (37 °C), and mixed on a vortex for 10 s. 2 ml of media/cells was removed and added to an equal volume of 100% FBS. This procedure was repeated 3 times. The dissociated cells were harvested by centrifugation (250 × g) for 10 min, resuspended in DMEM supplement with 20% FBS and 1% penicillin/streptomycin, and plated on glass coverslips (25 mm). Single cell calcium imaging was performed using the calcium-sensitive dye fura-2. The cells were washed with a physiological medium (KRH) containing NaCl (125 mM), KCl (5 mM), KH2PO4 (1.2 mM), MgSO4 (1.2 mM), CaCl2 (2 mM), glucose (6 mM), HEPES (25 mM), pH 7.4, and loaded with 2 µM fura-2 AM (Molecular Probes, Eugene, OR) for 50 min at 25 °C. Single cell recordings were collected using a Nikon Diaphot inverted microscope (Garden City, NY) coupled to a dual monochrometer system via a fiberoptic cable (Photon Technology International, South Brunswick, NJ). Fluorescence was detected using an intensified CCD camera (Dage-MTI, Inc., Michigan City, IN). Image frames were acquired every 1-8 s and analyzed using ImageMaster software (Photon Technology International).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of CCK-BRi4sv-- The novel CCK-BR splice variant was isolated from a cDNA library constructed with double-selected poly(A)+ mRNA from a freshly resected human colon cancer. The screening of 1 million plaques, using a random primed [32P]dATP-labeled CCK-BR cDNA probe, produced two cDNA clones of approximately 2.2 kb in length. The DNA sequences of the sense and antisense strands for each clone were determined and found to contain a single large open reading frame encoding a CCK-BR in which intron 4 was retained (CCK-BRi4sv). Retention of intron 4 did not cause a frameshift in the open reading frame or the introduction of a stop codon. Consequently, the amino acid sequence of CCK-BRi4sv, predicted from the cDNA, encodes a receptor protein with a 69-amino acid insertion in its putative third cytoplasmic domain (Fig. 1A). The amino acid sequence upstream and downstream of the intron 4 insertion site is identical to wild-type CCK-BR (CCK-BRwt) with the exception of one residue at position 64. A point mutation in the nucleotide sequence (T to C) resulted in a change in the amino acid residue at this position from an isoleucine in CCK-BRwt to a threonine residue in CCK-BRi4sv (Fig. 1A). Additionally, DNA sequence analysis revealed that retention of intron 4 was not due to mutations in either the 5' or 3' splice site consensus sequences (Fig. 1B).


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  		Fig. 1.   Human colorectal cancers express a novel CCK-BR splice variant. A, the predicted amino acid sequence and membrane topology of the novel CCK-BR splice variant. The gray circles with black letters designate sequences that are identical to CCK-BRwt. The black circles with white letters indicate the amino acid sequence encoded by intron 4. The cDNA library was constructed using the ZAP Express XL Library Construction Kit and mRNA isolated from tumor tissue of Patient 5 (Fig. 2C). B, a partial DNA sequence showing the intron 4 splice boundaries. The underlined sequences indicate the previously described 5' and 3' splice sites. The nucleotide numbering is based on the previously described CCK-BR cDNA sequence (GenBankTM accession number L08112) and is provided for reference.

CCK-BRi4sv Expression in Human Colorectal Cancers and Adenomatous Polyps-- To begin to assess the prevalence of CCK-BRi4sv expression in human colorectal cancers, we have screened DNase I-treated mRNA isolated from the resected cancers of eight patients (Table I) by reverse transcription-polymerase chain reaction (RT-PCR) assays. The oligonucleotide primers, used in the PCR reaction, were complementary to sequences found in the receptor's fifth and sixth transmembrane domains, flanking the third cytoplasmic loop. By using these primers, CCK-BRi4sv expression would be indicated by the presence of a 554-base pair (bp) PCR product (Fig. 2A), whereas the expression of CCK-BRwt would yield a 347-bp product (Fig. 2, A and B). A single PCR product, 554 bp in length, was detected in all eight cancers, indicating expression of CCK-BRi4sv (Fig. 2C, T1-T8). DNA sequence analysis and Southern blotting confirmed the presence of intron 4 sequence within the 554-bp PCR product (data not shown). After 40 cycles of amplification, we did not detect expression of either CCK-BRwt (347 bp) or CCK-BRi4sv (554 bp) in paired, normal colonic mucosa adjacent to the cancers (Fig. 2C, N1-N8). Because the CCK-BR gene contains three other introns (Fig. 2A), we also analyzed the RNA from tumor and normal tissue samples for the presence of these other introns. We did not detect any of the other three introns in the RNA samples from either cancers or normal mucosa (data not shown), indicating selective retention of intron 4. 

                              
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Table I
Summary of tumor site, TNM classification, and stage of the colorectal cancers


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  		Fig. 2.   Expression of CCK-BRi4sv* in human colorectal cancers and adenomatous polyps. A, diagram illustrating the splicing of the human CCK-BR gene to yield the novel CCK-BR splice variant (CCK-BRi4sv) RNA expressed in human colorectal cancers. For comparison, the previously characterized wild-type CCK-BR (CCK-BRwt) RNA is shown. Light gray rectangles (A-E) indicate exons. Black rectangles (1-4) indicate introns. The numbers in parentheses indicate the length of each exon and intron in base pairs (bp). Roman numerals I-VII indicate the relative locations of transmembrane domains. Arrows indicate the location of sense (S) and antisense (AS) oligonucleotide primers used in the PCR assays. B, ethidium bromide-stained agarose gel (1%) showing the 347-bp RT-PCR product from rat intestinal epithelial cells expressing recombinant human CCK-BRwt. The preparations of RNA and RT-PCR were performed as described under "Experimental Procedures." C, ethidium bromide-stained agarose gel (1%) showing products of RT-PCR assays using RNA from human colorectal tumors (T1-T8) and patient-matched, normal colonic mucosa (N1-N8). Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for RNA quality. D, RNase protection assay showing expression of CCK-BRi4sv in colorectal tumor (T) samples but not in normal (N) mucosa. Undigested probe is shown in the 2nd lane (Intron Probe). Yeast RNA was used to determine the level of nonspecific probe hybridization. The lane labeled M contains RNA markers of 100, 200, and 300 bp in length. E, CCK-BRi4sv is expressed in adenomatous polyps. Ethidium bromide-stained agarose gel (1%) shows products of RT-PCR using RNA from polyps of a patient with FAP.

Confirmation of the selective expression of CCK-BRi4sv in human colon cancers was further demonstrated by RNase protection assays. After treatment with RNase, a 185-bp fragment, corresponding to a portion of intron 4, was protected from digestion in tumor RNA samples isolated from patients 1, 5, 6, and 8 (Fig. 2D, T1, T5, T6, and T8). A protected band was not detected in matched RNA samples isolated from normal colonic mucosa (Fig. 2D, N1, N5, N6, and N8). CCK-BRi4sv expression was not detected by RNase protection assays in cancers from patients 2, 3, 4, and 7. These samples also showed a relatively faint 554-bp band by RT-PCR (Fig. 2C).

Adenomatous polyps are an early manifestation of dysregulated growth control of the colonic epithelium and a predisposing condition for colorectal cancer. To determine whether CCK-BRi4sv was expressed in these pre-malignant, hyperproliferative lesions, we analyzed four polyps from a patient with familial adenomatous polyposis (FAP). Like the cancers, expression of CCK-BRi4sv (554 bp), but not CCK-BRwt, was detected in all four polyps (Fig. 2E). Additional analyses of polyps from both FAP and non-FAP patients are required; however, these data do suggest a possible role for CCK-BRi4sv in early events leading to the development of colorectal cancer.

Radiolabeled Ligand Binding Studies-- To assess the effects of intron 4 retention and the subsequent 69 amino acid insertion on the binding and signaling properties of CCK-BRi4sv, we expressed either the splice variant or CCK-BRwt in mouse Balb3T3 fibroblasts. Although CCK-BRi4sv was the only form of CCK-BR detected in human colorectal cancers, examination of the cDNA revealed normal intron 4 splice site consensus sequences (Fig. 1B). Therefore, we reasoned that expression of CCK-BRi4sv in a non-tumorigenic cell line, such as Balb3T3 fibroblasts, would result in intron splicing and the generation of CCK-BRwt. To ensure that we were characterizing the properties of CCK-BRi4sv, and not CCK-BRwt, we introduced point mutations into the previously identified splice sites of intron 4 (24). The single nucleotide changes modified the splice consensus sequences without changing the amino acid composition of the protein (Fig. 3A). To confirm that the splice site mutant receptor (designated CCK-BRi4sv*) was expressed in transfected Balb3T3 cells, we performed RT-PCR analysis. As expected, before the splice sites were modified, a fraction of the CCK-BRi4sv transcript was converted to CCK-BRwt as indicated by the appearance of both the 554- and 347-bp PCR products (Fig. 3B, CCK-BRi4sv). After introduction of point mutations at the splice boundaries, only a 554-bp PCR product corresponding to the CCK-BRi4sv* transcript was detected (Fig. 3B, CCK-BRi4sv*). The effects of intron 4 retention on radiolabeled ligand binding and intracellular signaling were assessed using CCK-BRi4sv*.


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  		Fig. 3.   Effects of intron 4 retention on CCK-BRi4sv* ligand binding. A, diagram showing the location of the point mutations that were introduced at the 5' and 3' splice boundaries of intron 4 to convert CCK-BRi4sv to CCK-BRi4sv*. The single base changes were introduced using the Quick Change mutagenesis Kit (Stratagene, La Jolla, CA). B, RT-PCR using RNA from Balb3T3 cells expressing CCK-BRwt, CCK-BRi4sv, or CCK-BRi4sv*. The PCR products were resolved on a 1.5% agarose gel. A 1-kb DNA ladder (Life Technologies, Inc.) was use to determine the relative size of the PCR products. C and D, competition binding analyses comparing the binding properties of CCK-BRi4sv* and CCK-BRwt. The IC50, 95% confidence interval and R2 values calculated for competition of 125I-labeled G-17 binding by G-17, G-Gly, and L-60 to CCK-BRwt and CCK-BRi4sv* are summarized in Table II. Best-fit curves were generated using non-linear regression analyses for one or two sites with GraphPad Prism software (GraphPad, San Diego, CA). E, competition binding analyses using 125I-labeled G-Gly and Balb3T3 cells transfected with CCK-BRi4sv*. The IC50, 95% confidence intervals, and R2 values calculated for competition of 125I-labeled G-Gly binding by G-Gly were 312 (165-590 nM) and 0.93 nM, respectively. F, effects of the CCK-BRwt-selective antagonists, L-60 and CAM1028, on 125I-labeled G-Gly binding to CCK-BRi4sv*. Addition of unlabeled G-17 (1 µM) and G-Gly (1 µM) blocked binding of 125I-labeled G-Gly to CCK-BRi4sv*, whereas L-60 (1 µM) and CAM1028 (1 µM) had no effect. + indicates the presence of unlabeled competitor and - indicates no competitor.

Competition binding studies with Balb3T3 cells transiently transfected with CCK-BRi4sv* revealed two binding sites for G-17 as follows: one site that was competed by relatively low concentrations of G-17 (IC50 of 0.12 nM) and a second site that required higher concentrations (IC50 of 315 nM) (Fig. 3C) (Table II). By comparison, cells expressing CCK-BRwt exhibited a single binding site for G-17 (IC50 = 0.63 nM) (Fig. 3D). The non-amidated precursor of G-17, G-Gly, was about three times more effective at competing 125I-labeled G-17 bound to CCK-BRi4sv* (IC50 = 434 nM) than to CCK-BRwt (IC50 = 1.3 µM). The selective CCK-BRwt antagonist L365,260 (L-60) exhibited a similar potency for both receptors (IC50 = 2.3 nM for CCK-BRi4sv* and 3.6 nM for CCK-BRwt). However, L-60 competed only the 125I-labeled G-17 bound to the high affinity G-17 site on CCK-BRi4sv* (Fig. 3C). G-Gly binding to CCK-BRi4sv* was confirmed by direct competition of 125I-labeled G-Gly binding by G-Gly (IC50 = 312 nM) (Fig. 3E). Radiolabeled G-Gly binding to CCK-BRi4sv* was also insensitive to inhibition by the selective antagonists L-60 (1 µM) and CAM1028 (1 µM) (Fig. 3F), suggesting that G-Gly binds to the low affinity G-17-binding site on CCK-BRi4sv*.

                              
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Table II
Summary of competition binding studies

Effects of CCK-BRi4sv* Expression on Intracellular Ca2+ Signaling-- Binding of G-17 to CCK-Bwt is coupled, in part, to the mobilization of intracellular Ca2+ through the activation of heterotrimeric G proteins of the Gq subfamily and the phospholipase C-dependent generation of inositol 1,4,5-trisphosphate (IP3) (10). By using single-cell [Ca2+]i imaging, we found that collagenase-dissociated colorectal cancer cells exhibited both spontaneous, non-synchronous, and oscillatory changes in [Ca2+]i as well as G-17-stimulated increases in [Ca2+]i (Fig. 4A). Balb3T3 cells transfected with CCK-BRi4sv* exhibited a similar pattern of ligand-independent and G-17-dependent increases in [Ca2+]i (Fig. 4B). In contrast, cells expressing either CCK-BRwt or the empty expression vector, pcDNA3.1, did not exhibit spontaneous [Ca2+]i oscillation (Fig. 4, C and D); however, a G-17-stimulated response was evoked in cells expressing CCK-BRwt (Fig. 4C). Because increases in GPCR signaling, in the absence of agonist, can result from receptor overexpression (25), we assessed the level of receptor expression using radiolabeled ligand binding. These studies showed similar levels of 125I-labeled G-17 binding to both CCK-BRi4sv* and CCK-BRwt (Fig. 4E), demonstrating that the spontaneous, ligand-independent increases in [Ca2+]i observed in CCK-BRi4sv* expressing cells were an intrinsic property of the receptor and not due to receptor overexpression.


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  		Fig. 4.   Colon cancer cells and Balb3T3 cells expressing CCK-BRi4sv* exhibit both constitutive (ligand-independent) and G-17-stimulated increases in [Ca2+]i. A, primary cultures of cells isolated from human colorectal tumors exhibit both spontaneous and G-17-stimulated increases in [Ca2+]i. The change in [Ca2+]i is expressed as the ratio of fura-2 fluorescence at 340/380 nm. The tracings from 5 to 10 individual cells are shown. The black bar indicates the period of time the cells were exposed to indicated ligands. B, Balb3T3 cells expressing CCK-BRi4sv* exhibited both spontaneous and G-17-stimulated increases in [Ca2+]i. C, Balb3T3 cells expressing CCK-BRwt showed a G-17-stimulated increase in [Ca2+]i but did not exhibit spontaneous fluctuations in [Ca2+]i. D, cells transfected with the empty expression vector, pcDNA3.1 (Invitrogen, Carlsbad, CA), did not displace spontaneous or G-17-stimulated [Ca2+]i activity. E, radiolabeled G-17 binding assays demonstrated that both CCK-BRi4sv* and CCK-BRwt cells expressed similar levels of receptors (TB, total binding; NS, nonspecific binding). F, treatment of colon cancer cells with the CCK-BR-selective antagonist, CAM1028, blocked a G-17-induced [Ca2+]i; however, CAM1028 had no effect on the spontaneous, ligand-independent [Ca2+]i activity. G and H, Balb3T3 cells expressing CCK-BRi4sv* were treated with G-17 in the presence and absence of CAM1028.

To determine whether the high affinity G-17-binding site on CCK-BRi4sv* mediated the agonist-stimulated increase in [Ca2+]i, we examined the effects of the CCK-BR-selective antagonist, CAM1028, on the G-17-stimulated Ca2+ responses. CAM1028 blocked the G-17-stimulated increase in [Ca2+]i in primary cultures of human colorectal cancer cells (Fig. 4F) but did not affect the spontaneous Ca2+ activity. Similarly, treatment of Balb3T3 cells expressing CCK-BRi4sv* with G-17 induced a synchronized increase in [Ca2+]i (Fig. 4G) which was blocked by CAM1028 (Fig. 4H). The antagonist, however, did not inhibit the spontaneous, ligand-independent [Ca2+]i oscillations associated with CCK-BRi4sv* expression.

Effect of CCK-BRi4sv* Expression on Cell Proliferation-- To determine the effect of CCK-BRi4sv* expression on cell proliferation, we performed cell counting experiments on both stably transfected Balb3T3 cell lines and transiently transfected rat NRK-49F cells. Fig. 5A shows the results obtained from the stable cell lines used in Fig. 4. In the absence of G-17 stimulation, there was a significant increase in the number of cells per culture in cells stably transfected with CCK-BRi4sv* at 24, 48, and 72 h after plating, compared with either cells stably transfected with CCK-BRwt or the expression vector, pcDNA3.1. By 72 h, an approximately 2.5-fold increase in cell number was observed (Fig. 5A, CCK-BRi4sv*; 20,940 ± 926 versus CCK-BRwt; 7,947 ± 266 and pcDNA3.1; 8,773 ± 384). To determine the effect of G-17 and G-Gly on cell growth, the three cell lines were treated with either 100 nM G-17 or 1 µM G-Gly for 24, 48, and 72 h. We observed no additional effect of peptide treatment on cell growth (data not shown). The trophic effects of CCK-BRi4sv* expression were confirmed using rat NRK-49F cells transiently transfected with either CCK-BRi4sv*, CCK-BRwt, or pcDNA3.1. One day after transfection, the NRK-49F were replated, and single cells were identified. Three days later, the number of cells arising from the previously identified single cell was determined. We observed an approximately 2-fold increase in the number of cells per cell cluster in CCK-BRi4sv*-expressing cells compared with cells transfected with either CCK-BRwt or the empty vector, pcDNA3.1 (Fig. 5, B and C) (CCK-BRi4sv*, 28 ± 2.4; CCK-BRwt, 17 ± 2.0; pcDNA3.1, 14 ± 1.9). Together these data demonstrate that expression of CCK-BRi4sv* alone, in the absence of agonist stimulation, is sufficient to stimulate cell proliferation.


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  		Fig. 5.   Expression of CCK-BRi4sv* in rodent fibroblasts increases their growth rate compared with cells expressing CCK-BRwt or the empty expression vector, pcDNA3.1. A, the Balb3T3 cell lines shown in Fig. 4, expressing either CCK-BRi4sv*, CCK-BRwt, or the empty expression vector, were plated on 6-well plates at a density of 10,000 cells per well in DMEM supplemented with 5% heat-inactivated FBS. After 24, 48, and 72 h in culture, the cells were trypsinized and counted in a Coulter cell counter. B, rat NRK-49F (ATCC) were transiently transfected with either CCK-BRi4sv*, CCK-BRwt, or pcDNA3.1 as described (28). Twenty four hours after transfection, the cells were trypsinized and replated at a density of 5,000 cells/100-mm plate in DMEM supplemented with 10% FBS. Six to 8 h after replating, individual cells were identified. After 3 days in culture, the cells, resulting from a previously identified single cell, were counted using Nomarski optics. The graph shows the mean number of cells per clusters ± S.E. (n = 50 clusters/plate) from two separate experiments performed in triplicate. Statistical differences between CCK-BRi4sv*, CCK-BRwt, and pcDNA3.1 cells were determined with one-way analysis of variance; dagger , p < 0.0001. C, photomicrograph (× 10 phase) of representative cell clusters of NRK-49F cells, transfected with either pcDNA3.1, CCK-BRi4sv*, or CCK-BRwt.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The possibility that the gastrointestinal peptide hormone, gastrin, and it biosynthetic precursor, G-Gly, play a role in the development of colorectal cancers has aroused considerable interest over the past several years. Most colorectal cancers and derived cell lines produce amidated G-17 and/or G-Gly (26, 27). Also, many colorectal cancers exhibit a proliferative response upon application of exogenous peptides. Together these observations have led to the hypothesis that G-17 and its non-amidated precursors act as autocrine and/or paracrine growth factors to stimulate colorectal cancer cell proliferation. In this model, G-17 and/or G-Gly are released from cancer cells into the interstitium where they bind to specific cell-surface receptors on the same or adjacent cells and stimulate proliferation (18). Here we have reported the isolation and partial characterization of a novel splice variant of CCK-BR, designated CCK-BRi4sv, that stimulates cell growth in a G-17-independent manner. Our data show that colorectal cancers and adenomatous polyps, but not the normal colonic mucosa, express CCK-BRi4sv and that cells expressing this receptor exhibit spontaneous, oscillatory increases in [Ca2+]i in the absence of agonist stimulation.

The constitutive, ligand independent activation of pathways regulating [Ca2+]i and cell proliferation exhibited by CCK-BRi4sv may contribute to its potential pathophysiological role in colorectal cancers. Recently, other GPCRs exhibiting constitutive activation of intracellular signaling pathways have been identified as causative factors in several human diseases including some cancers (28, 29). The human Kaposi's sarcoma-associated herpesvirus encodes a GPCR, with homology to the human interleukin-8 receptor, which constitutively activates the IP3/[Ca2+]i pathway and, when transfected into rat NRK-49F cells, increases their rate of growth in the absence of agonist stimulation (29). We have shown that CCK-BRi4sv* can stimulate the rate of cell growth in the absence of agonist stimulation which suggests that, like Kaposi's sarcoma-associated herpesvirus, constitutive action of the IP3dependent/[Ca2+]i pathway may be involved in the regulation of cell proliferation. Several other G protein-coupled receptors that signal through Galpha q/IP3/[Ca2+]i pathway have been shown to stimulate DNA synthesis and induce a transformed-like phenotype in fibroblasts. These include the serotonin 1c, muscarinic acetylcholine m1, m3, and m5, and the alpha 1b-adrenergic receptors (30-33). However, unlike CCK-BRi4sv and Kaposi's sarcoma-associated herpesvirus, the stimulation of DNA synthesis and transformation, by these other receptors, required the continued presence of agonist.

Various types of reconstitution studies and experiments with natural membrane systems have shown that GPCRs can spontaneously activate G proteins in the absence of agonist (34, 35). A thermodynamic scheme for receptor-G protein interactions has been presented (36, 37) as an extension of the well known ternary complex model of receptor-effector interactions. In this model, a receptor, in the absence of ligand, can exist in so-called inactive (Ri) and active (Ra) states with respect to its predisposition to associate spontaneously with G protein. The activated receptor (Ra) can spontaneously form a complex with G protein (Ra·G protein). Our data support the hypothesis that the structural alterations associated with the addition of 69 amino acid residues to the third intracellular loop domain of CCK-BRi4sv cause it to favor the Ra conformation and, therefore, increase its predisposition to spontaneous coupling to G protein. The consequences of the increased spontaneous coupling to G protein is reflected by the transient elevations in [Ca2+]i. Another important aspect of this model, which is also supported by our data, is that an agonist has two different binding states of the receptor to choose from, namely Ri and Ra (and Ra·G protein). CCK-BRi4sv* expressed in Balb3T3 cells clearly exhibits two binding states for G-17. Additionally, this model predicts that a ligand that destabilizes Ra·G protein will act as an inverse agonist and inhibit spontaneous signaling by the receptor. Inverse agonists for CCK-BRi4sv could prove to be useful therapeutic agents.

The similarities in both the pattern and pharmacology of the [Ca2+]i responses observed in Balb3T3 cells transfected with CCK-BRi4sv* and primary cultures of collagenase-dissociated colorectal cancer cells strongly suggest that CCK-BRi4sv is the mediator of the response in the latter. Although the identification of CCK-BRi4sv does not preclude the existence of other G-17/G-Gly receptors on human colorectal cancers, its selective pattern of expression, constitutive (ligand-independent) activity, and growth-promoting effects provides compelling evidence for a significant role of CCK-BRi4sv in colorectal carcinogenesis. The unique properties of CCK-BRi4sv suggest that it may stimulate colorectal cancer cell proliferation though a gastrin-independent mechanism and make it an attractive target for the development of novel drugs such as inverse agonists, which, in the future, may provide more effective adjuvant therapies for this devastating disease.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01DK48345 (to C. M. T.) and an American Cancer Society institutional grant (to M. R. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF239668.

To whom correspondence should be addressed: Dept. of Surgery, the University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0722. Tel.: 409-772-1845; Fax: 409-747-7383; E-mail: mhellmic@utmb.edu.

Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M005754200

    ABBREVIATIONS

The abbreviations used are: CCK-BR, cholecystokinin B/gastrin receptor; [Ca2+]i, intracellular free Ca2+; GPCRs, G protein-coupled receptors; kb, kilobase pair; PCR, polymerase chain reaction; bp, base pair; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; RT-PCR, reverse transcription-polymerase chain reaction; IP3, inositol 1,4,5-trisphosphate; FAP, familial adenomatous polyposis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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